Invivo agents comprising cationic metal chelators with acidic saccharides and glycosaminoglycans

ABSTRACT

This application concerns novel agents comprising cationic or chemically basic metal chelators in association with hydrophilic carriers of anionic or chemically acidic saccharides, sulfatoids and glycosaminoglycans. In certain embodiments, the agents comprise metals and metal ions. Covalent and non-covalent chemical and physical means are described for stabilizing the binding of the metal chelators to the carriers. Novel non-covalently bound compositions are described which give uniquely high payloads and ratio of metal chelator to carrier, ranging from a low of about 15% metal chelator by weight, to a characteristic range of 70% to 90% metal chelator by weight. Specific embodiments are described comprising deferoxamine, ferrioxamine, iron-basic porphine, iron-triethylenetetraamine, gadolinium DTPA-lysine, gadolinium DOTA-lysine and gadolinium with basic derivatives of porphyrins, porphines, expanded porphyrins, Texaphyrins and sapphyrins as the basic or cationic metal chelators, which are in turn, bound to acidic or anionic carriers, including one or more of acidic or anionic saccharides, and including sulfated sucrose, pentosan polysulfate, dermatan sulfate, oversulfated dermatan sulfate, chondroitin sulfate, oversulfated chondroitin sulfate, heparan sulfate, beef heparin, porcine heparin, non-anticoagulant heparins, and other native and modified acidic saccharides and glycosaminoglycans. 
     Also disclosed are methods of enhancing in vivo imgages arising from induced magnetic resonance signals, methods of enhancing in vivo images in conjunction with ultrasound or X-rays and methods of obtaining in vivo body images utilizing radioisotope containing agents. Methods of treating vasular disease are also disclosed.

This is a continuation-in-part of U.S. Ser. No. 07/880,660 filed May 8,1992 abandoned, U.S. Ser. No. 07/863,595 filed Apr. 3, 1992, now U.S.Pat. No. 5,214,661 and U.S. Ser. No. 07/642,033 filed Jan. 1, 1991, nowU.S. Pat. No. 5,336,762, all of which are incorporated by referenceherein.

The present invention describes novel compositions, agents and methodsof in vivo use which give improved selectivity, efficacy, uptakemechanism and kinetic-spatial profiles at sites of disease. It furtherdescribes compositions, agents and methods of use for improvedselectivity, sensitivity, uptake mechanism and kinetic-spatial profilesof biomedical imaging, image contrast and spectral enhancement at sitesof disease, including but not limited to magnetic resonance image (MRI)contrast enhancement. Novel compositions are prepared by (a) uniquenon-covalent chemical binding, further enhanced by (b) physicalstabilization. Other compositions are prepared by covalent chemicalbinding. Binding is of cationic or chemically basic metal chelators tocarriers comprising anionic or chemically acidic saccharides, sulfatoidsand glycosaminoglycans, typically and advantageously of a hydrophilic oressentially completely hydrophilic nature. Binding of the active andcarrier may also be by a combination of non-covalent, physical, andcovalent means. Non-covalent binding can be carried out by meansincluding but not limited to admixing cationic or basic metal chelatorsat appropriate ratios with anionic or acidic saccharide carriers,thereby forming solution-state and dry-state paired-ion salts, basedprincipally on electrostatic binding of cationic (basic) group or groupsof the metal chelator to anionic (acidic) group or groups of the acidiccarrier. Such binding may be further stabilized by hydrogen bonds andphysical factors, including but not limited to concentration, viscosity,and various means of drying, including lyophilization.

Carrier substances useful in this invention may include, but are notlimited to natural and synthetic, native and modified, anionic or acidicsaccharides, disaccharides, oligosaccharides, polysaccharides andglycosaminoglycans (GAGs). It will be apparent to those skilled in theart that a wide variety of additional biologically compatible,water-soluble and water dispersable, anionic carrier substances can alsobe used. Due to an absence of water-diffusion barriers, favorableinitial biodistribution and multivalent site-binding properties,oligomeric and polymeric, hydrophilic and substantially completelyhydrophilic carrier substances are included among the preferred carriersfor agents to be used for paramagnetic, T1-Type, selective MRI contrastof tumors, cardiovascular infarcts and other T1-Type MRI contrast uses.However, it will be apparent to those skilled in the art that amphotericand hydrophobic carriers may be favored for certain biomedical imagingapplications and therapeutic applications. Metal chelators useful inthis invention include those which contain cationic, basic andbasic-amine groups and which chelate metals and metal ions, transitionelements and ions, and lanthanide series elements and ions. It will beapparent to those skilled in the art that essentially any single atomicelement or ion amenable to chelation by a cationic, basic andamine-containing chelator, may also be useful in this invention.

For purposes of this invention, a cationic or basic metal chelator isdefined and further distinguished from a metal-ion complex as follows: acationic or basic metal chelator comprises an organic, covalent,bridge-ligand molecule, capable of partly or entirely surrounding asingle metal atom or ion, wherein the resulting formation constant ofchelator for appropriate metal or ion is at least about 10¹⁴. A chelatoris further defined as cationic or basic if it or its functional group orgroups which confer the cationic or basic property, and which includebut are not limited to an amine or amines, is (are) completely oressentially completely electrophilic, positively charged or protonatedat a typical pH employed for formulation. A formulation pH ischaracteristically selected to closely bracket the range of physiologicpH present in mammalian vertebrates. This typically includes, but is notlimited to a pH in the range of pH 5 to 8. Amines may include primary,secondary, tertiary or quaternary amines or combinations thereof on themetal chelator. Herein, and as specified, a hydrophilic carrier isdefined as a substance which is water soluble, partitions into the waterphase of aqueous-organic solvent mixtures, or forms a translucentaqueous solution, complex, aggregate, or particulate dispersion underthe conditions employed for formulation. A carrier is further defined asbeing anionic or acidic if it is completely or nearly completelynucleophilic, or if its functional group or groups capable ofinteracting with cationic, basic or amine metal chelators, is (are)completely or nearly completely negatively charged, anionic or ionizedat the pH employed for formulation. Such anionic and acidic groupsinclude, but are not limited to sulfates, phosphates and carboxylates,or combinations thereof on the carrier.

Novel agent compositions include, but are not limited to the classes ofcationic or basic, typically basic-amine metal chelator actives, ormetal chelator actives including the chelated metal or metal ion,wherein these actives are further bound to anionic and acidic carrierscomprising natural, synthetic or synthetic carriers, including but notlimited to hydrophilic anionic or acidic, natural or synthetic, native,modified, derivatized and fragmented, anionic or acidic saccharides,oligosaccharides, polysaccharides, sulfatoids, and glycosaminoglycans(GAGs).

Anionic and acidic saccharide and glycosaminoglycan carriers may containmonomeric units comprising glucose, glucuronic acid, iduronic acid,glucosamine, galactose, galactosamine, xylose, mannose, fucose, sialicacid, pentose, and other naturally occurring, semi-synthetic orsynthetic monosaccharides or chemical derivatives thereof, comprisingamine, sulfate, carboxylate, sialyl, phosphate, hydroxyl or other sidegroups. Glycosaminoglycans (GAGs) comprise essentially the carbohydrateportions of cell-surface and tissue matrix proteoglycans. They arederived from naturally occurring proteoglycans by chemical separationand extraction; and in certain instances, by enzymatic means Lindahl etal. (1978), incorporated herein by reference!. They include, but are notlimited to those of the following types: heparin, heparan sulfate,dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, keratansulfate, syndecan, and hyaluronate, and over-sulfated, hyper-sulfated,and other chemical derivatives thereof, as described further below.

Strongly acidic glycosaminoglycans include all of those classes listedjust above, except for hyaluronate, which contains only the more weaklyacidic carboxylate groups and not sulfate groups. Natural sources ofglycosaminoglycans include, but are not limited to: pig and beefintestinal mucosa, lung, spleen, pancreas, and a variety of other solidand parenchymal organs and tissues.

Sulfatoids comprise a second class of sulfated saccharide substanceswhich are derived principally but not exclusively from bacterial andnon-mammalian sources. Sulfatoids are typically of shorter chain lengthand lower molecular weight than glycosaminoglycans, but may besynthetically modified to give (a) longer chain lengths, (b) increasedsulfation per unit saccharide, (c) various other chemical side groups,or (c) other properties favorable to the desired ligand-binding propertyand site-selective binding, uptake and accumulation property (orproperties) in vivo. Sucrose and other short-chain oligosaccharides maybe obtained from natural and synthetic sources.

These oligosaccharides can be rendered anionic or acidic by chemical orenzymatic derivatization with carboxylate, phosphate, sulfate or silylside groups, or combinations thereof, at substitution ratios of up toabout eight anionic or acidic substituent groups per disaccharide unit.Modified glycosaminoglycans may be derived from any of the types andsources of native glycosaminoglycans described above, and include: (1)glycosaminoglycan fragments, further defined as glycosaminoglycans withchain lengths made shorter than the parental material as isolateddirectly from natural sources by standard ion-exchange separation andsolvent fractionation methods; (2) glycosaminoglycans chemicallymodified to decrease their anticoagulant activities, thereby giving"non-anticoagulant" (NAC) GAGs, prepared typically but not exclusivelyby (a) periodate oxidation followed by borohydride reduction; (b)partial or complete desulfation; and (c) formation of non-covalentdivalent or trivalent counterion salts, principally including but notlimited to salts of the more highly acidic sulfate functional groups,with principally but not exclusively: calcium, magnesium, manganese,iron, gadolinium and aluminum ions.

For purposes of this invention, a special class of such salts includesthose salts formed by electrostatic or paired-ion association betweenthe acidic or sulfate groups of acidic saccharide or glycosaminoglycancarrier, and the basic or cationic group or groups of the metal chelatoror metal chelator including metal, as described above. Derivatizedacidic saccharides and glycosaminoglycans are typically prepared byderivatization of various chemical side groups to various sites on thesaccharide units. This may be performed by chemical or enzymatic means.

Enzymatic means are used in certain instances where highly selectivederivatization is desired. Resulting chemical and enzymatic derivativesinclude, but are not limited to acidic saccharides andglycosaminoglycans derivatized by: (1) esterification of (a) carboxylategroups, (b) hydroxyl groups, and (c) sulfate groups; (2) oversulfationby nonselective chemical or selective enzymatic means; (3) acetylation,and (4) formation of various other ligand derivatives, including but notlimited to (a) addition of sialyl side groups, (b) addition of fucosylside groups, and (c) treatment with various carbodiimide, anhydride andisothiocyanate linking groups, and (d) addition of various otherligands.

If and when present, sulfate and sialyl side groups may be present atany compatible position of saccharide monomer, and on any compatibleposition of glycosaminoglycan monomers Lindahl et al. (1978),incorporated herein by reference!. Certain of the resulting derivatizedacidic saccharides and glycosaminoglycans may have desired alterationsof anticoagulant activities, site-localization patterns, clearance andother biological properties. As one example of this relationship betweencertain classes of glycosaminoglycans and biological properties,dermatan sulfates with a native sulfate/carboxylate ratio of 1:1, arereported to have relatively low binding to normal endothelial cells,avoid displacement of endogenous heparan sulfate from endothelial-cellsurfaces, have relatively high selectivity to induced endothelia atsites of disease, including thrombus, and have rapid plasma clearance,principally by the renal route; whereas heparins and oversulfateddermatan sulfates with higher sulfate/carboxylate ratios of between 2:1and 3.7:1, are reported to have relatively higher binding for bothnormal and induced endothelia, to displace relatively more endogenousendothelial heparan sulfate, and to clear more slowly than dermatansBoneu et al. (1992), incorporated herein by reference!.

In a special case unique to the present invention, derivatization of theacidic saccharide and glycosaminoglycan carriers may be accompanied bythe basic metal chelator itself. Although the general classes ofcarriers described above are particularly suitable to the presentinvention, it will be apparent to those skilled in the art that a widevariety of additional native, derivatized and otherwise modifiedcarriers and physical formulations thereof, may be particularly suitablefor various applications of this invention. As one representativeexample, the source and type of glycosaminoglycans, its chain length andsulfate/carboxylate ratio can be optimized to (1) provide optimalformulation characteristics in combination with different small andmacromolecular diagnostic agents and drugs; (2) modulate carrierlocalization on diseased versus normal endothelium; (3) minimizedose-related side effects; (4) optimize clearance rates and routes ofthe carrier and bound diagnostic and therapeutic actives.

Non-covalent formulations of active and carrier afford markedly higheractive-to-carrier ratios than those possible for covalent chemicalconjugates. In the present invention, non-covalent binding affords aminimum of 15% active to total agent by weight active/(active+carrier),w/w!; typically greater than about 30% (w/w); preferably at least about50% (w/w); and frequently between about 70-99% (w/w). Covalent bindingcharacteristically limits the percent active to (a) less than about 12%for non-protein small and polymeric carriers, (b) less than about 7% forpeptide and protein carriers, including antibodies, and (c) less thanabout 0.5-2.0% for antibody fragments. This limitation is based on thenumber of functional groups available on carrier molecules which areuseful in agent formulation and in vivo site localization.

It will be apparent to those skilled in the art that covalentactive-carrier agent compositions of low substitution ratio may beuseful for certain in vivo applications of typically narrow range, andthat non-covalent active-carrier agent compositions of high substitutionratio may be useful for other in vivo applications of typically broaderrange. Generally, but not exclusively, covalent agents may be useful forradionuclide imaging or therapeutic applications in which only lowtotal-body doses are needed, clearance of the non-targeted dose fractiondoes not cause undue toxicity, and high conjugate stability is required.Generally, but not exclusively, non-covalent agents may be particularlyuseful for the majority of diagnostic imaging applications and certainhigh-dose therapeutic applications, for which high total-body andsite-localized doses are needed, and rapid clearance of thenon-localized fraction of administered agent is desired in order toaccelerate plasma clearance and to achieve low background levels forpurposes of maximizing image contrast and minimizing systemic toxicity.

Rapid clearance is preferentially conferred by non-covalent physicalformulations due to their capacity to give controlled dissociation orrelease of the active from the carrier. Such controlled release allowsthe diagnostic or therapeutic active, to dissociate from its carrier ata programmed rate which is consistent with rapid site localization of asignificant fraction of the total administered dose. In instances wherethe carrier is polymeric and hence clears more slowly, this selectivelyaccelerates clearance of the active.

It will be apparent to those skilled in the art that such controlledrelease can also be achieved for actives which are chemically conjugatedto their carriers via chemical linkers, including peptide linkers, whichare susceptible to cleavage by body enzymes. However, this latter meansof facilitated clearance: (a) gives much longer clearance times than dophysical formulations, (b) depends on endogenous enzyme levels andinhibitors which typically differ from subject to subject, from healthto disease, and from one stage of disease to another. Hence, physicalformulations have substantial advantages over chemical conjugates fromthe standpoints of both (a) high payload, and (b) accelerated clearance.

These properties of the present formulations represent additionalsubstantial improvements over prior, non-selective and covalentlyconjugated active-carrier agents. The resulting agents are broadlyuseful for: (a) MRI contrast and spectral enhancement, Ultrasoundcontrast enhancement, and X-Ray contrast enhancement, where relativelyhigh administered doses may be favored or required; (b) Nuclear Medicalor Radionuclide imaging and therapy, where enhanced clearance of thenon-targeted dose may be favored or required: and (c) certain high-dose,extended-release or sustained-effect therapy may be favored or required.Such therapeutic agents include but are not limited to those useful at abroad variety of organ sites and medical indications, for the treatmentof: (a) acute vascular ischemia, acute infarct, acute vascular damage,shock, hypotension, restenosis, proliferation of neo-vessel, parenchymalcells or other pathological proliferations; and (b) the followingclasses of disease: vascular, parenchymal, mesenchymal, endothelial,smooth muscle, striated muscle, adventitial, immune, inflammatory,bacterial, fungal, viral, degenerative, neoplastic, genetic andenzymatic.

MRI contrast enhancement is one important indication for which highpayload and controlled release of active are important unique advantagesin addition to site selective localization (see below). A still furtheradvantage is the hydrophilic form of carrier, which maximizes proximalwater diffusion and binding of the paramagnetic active. This lastproperty is required for optimal efficacy and minimal toxicity, becauseMRI paramagnetic T1-Type contrast agents require unimpeded waterdiffusion to within a very short distance of the localized metal ion inorder to achieve effective paramagnetic relaxation and T1 contrast.Additionally, MRI image instrumentation and image acquisition areinherently both of low sensitivity; and these limitations remain even atthe highest clinically acceptable field strengths and gradients and atthe optimal radiofrequency pulse sequences.

MRI paramagnetic agents have been prepared as stabilized liposomes,which contain up to about 22% of active (w/w). However, theirhydrophobic lipid bilayers markedly impede water diffusion into theliposome core active. This decreases their efficacy per unit doserelative to the hydrophilic controlled-release carriers of the presentinvention. There is an additional disadvantage of the reported MRIliposome formulations as follows: aside from localization in normalliver and reticuloendothelial-phagocytic organs, they have notdemonstrated effective site-localization at sites of tumors, infarctsand other focal pathology within tissue sites.

For purposes of this invention, metal ions generally useful forchelation in paramagnetic T1-Type MRI contrast agent compositions anduses may include divalent and trivalent cations selected from the groupconsisting of: iron, manganese, chromium, copper, nickel, gadolinium,erbium, europium, dysprosium and holmium. Chelated metal ions generallyuseful for radionuclide imaging and compositions and uses, and inradiotherapeutic compositions and uses, may include metals selected fromthe group of: gallium, germanium, cobalt, calcium, rubidium, yttrium,technetium, ruthenium, rhenium, indium, iridium, platinum, thallium andsamarium. Metal ions useful in neutron-capture radiation therapy mayinclude boron and others with large nuclear cross sections. Metal ionsuseful in Ultrasound contrast and X-Ray contrast compositions and usesmay, provided they achieve adequate site concentrations, include any ofthe metal ions listed above, and in particular, may include metal ionsof atomic number at least equal to that of iron.

For purposes of this invention, agents for therapeutic composition anduses in chelating internal body iron, copper or both, in order to makethese metals unavailable locally (1) which are typically required forneovascularization, or (2) which cause and amplify local tissue injuryLevine (1993), incorporated herein by reference!, include the carrierwith basic metal chelator in one or both of the following forms: (a)carrier plus chelator without metal ion; and (b) carrier plus chelatorwith metal ion added and chelated in the composition at a formationconstant lower or equal to that of the internal body metal which is tobe chelated by metal ion exchange into the respective basic metalchelator of the composition (see below). Such weakly chelated metal ionsof the composition may include one selected from the group of: calcium,manganese, magnesium, chromium, copper, zinc, nickel, iron, aluminum,cobalt, gadolinium or other exchangeable ion. Metal ions useful forinclusion in compositions for other therapeutic uses may include thedivalent and trivalent cations selected from the group of magnesium,manganese, chromium, zinc and calcium, iron, copper and aluminum. Itwill be obvious to those skilled in the art that various one of thepreceding metal ions can be used in combination with basic metalchelators, for alternative indications than those specified above, andthat metal ions other than those listed above may, under certainconditions, be useful in the uses and indications listed above.

The compositions described in this invention give surprising andunexpected improvements of performance and use which include:

(1) retained high association of active plus carrier during in vitrodialysis and in vivo targeting;

(2) selective binding of the active plus carrier to induced endotheliaat sites of disease;

(3) following intravenous administration, very rapid (2-7 min)localization at the diseased site, due to rapid selective endothelialbinding, envelopment and extravasation of the carrier plus metalchelator across disease-induced endothelia (including histologicallynon-porous endothelia);

(4) widespread uptake throughout the diseased tissue site;

(5) sustained retention (multiple hours to days) within the diseasedsite in combination with

(6) rapid plasma clearance (minutes) of the non-targeted fraction;

(7) moderately slow, polymeric diffusion rates within the diseasedtissue matrix, allowing differentiation of functional tissue subregionsbased on differences in perfusion of viable and non-viable subregions;

(8) capacity to selectively image solid tumors or acute vascular andmyocardial infarcts at body sites, as well as at brain and centralnervous system sites, with substantially improved selectivity,sensitivity, improved delineation of tumor and infarct boundaries atboth very short and prolonged post-injection intervals, and improveddetection of small tumor metastases, including those at liver and lungsites.

Diagnostic and drug enhancement can be made to occur by a number ofmechanisms, the principal ones being:

1. Effective TARGETING to tissue sites of disease;

2. STABILIZATION during both storage and plasma transit;

3. Prolonged RETENTION at the site of disease, giving a markedlyincreased area under the curve at the tissue site;

4. RAPID CLEARANCE of the non-TARGETED fraction, thereby reducingbackground signal in imaging applications and reducing normal organexposure and systemic toxicity in therapeutic applications.

Five further significant advantages of the present compositions and usesare:

1. Simple formulations of active and carrier;

2. Stabilization of diagnostic and therapeutic actives on the shelf andduring plasma transit;

3. Rapid site localization and sustained site retention;

4. Rapid clearance of the non-targeted fraction;

5. Availability of low toxicity carbohydrate carriers from naturalsources and, where needed, modification or derivatization bystraightforward synthetic means.

Acidic or anionic saccharides and glycosaminoglycans have uniquemechanisms of site localization and retention in vivo. They bind to thebody's endothelial determinants which are selectively induced on themicrovascular barrier by underlying tissue disease. Previous approachesto site targeting were directed at antigenic determinants. However,because these determinants are typically located in sequestered siteswithin the tissues, in other words at sites across the endothelialbarrier and not within the bloodstream and not on its endothelialsurface, carriers and agents injected into the bloodstream had noeffective means to recognize and localize in the region of these targetantigens. Stated another way, previous approaches ignored the majorproblem of inappropriate carrier distribution which resulted from itsfailure to recognize the vascular access codes required for efficientextravasation at disease sites. Hence, these carriers failed toeffectively load the relevant tissue sites with effective concentrationsof their bound actives.

The biological address of a disease site can be viewed in a fashionsimilar to that of a postal address, wherein effective carriersubstances must (1) first recognize the "state" address of the signalendothelium induced by proximal tissue disease; (2) next extravasate andload the "city" address of the extracellular tissue matrix with locallyeffective doses of the diagnostic and therapeutic actives; and (3)finally bind and load the "street" address of the target cells andantigens. Previous approaches to site delivery have attempted torecognize the "street" address without first recognizing the "state" and"city" addresses.

The reason that acidic saccharide and glycosaminoglycan systems worksubstantially better than previous antigen-recognition approaches, isthat they recognize the newly induced signals which the body uses toattract and target white blood cells into sites of tissue disease. Whendisease strikes at a local site, it initiates a cascade of localmediators within the tissue matrix and at the endothelial-bloodinterface which signal the blood cells and central body systems thatinflammatory and immune cells are required within the tissue site. Thesemediators include cytokines, chemoattractants, cytotaxins, inducedcell-surface adhesions, selectins and integrins, and varioustissue-derived and blood-borne, soluble and cell-surface procoagulants.White cell accumulation begins within minutes and continues over days toweeks, depending on the nature, severity and persistence of localdisease and the continued generation of tissue mediators andtrans-endothelial signals.

As has now been reported and reviewed in detail Ranney (1990); Ranney(1992); Bevilaqua et al. (1993); Bevilacqua et al. (1993); Travis(1993); Sharon et al. (1993), all incorporated herein by reference!,tumors, infarcts, infections, inflammatory diseases, vascular disorders,and other focal diseases, characteristically induce the release of suchhost mediators, or cytokines, from resident macrophages and local tissuematrix. In certain diseases, alien mediators such as bacteriallipopolysaccharides (LPS), viral RNA, and tumor-derived inducers,including EMAP II, and chemoattractants may also be released. Althoughadditional mediators remain to be elucidated, the principal ones havenow been defined and include: interleukin 1 (IL-1), tumor necrosisfactor (TNF), transforming growth factor beta (TGF-beta),Lipopolysaccharide (LPS), single and double stranded nucleotides,various interferons, monocyte chemoattractant protein (MCP), interleukin8 (IL-8), interleukin 3 (IL-3), interleukin 6 (IL-6), tumor-derivedinducers and chemoattractant peptides (as above), various prostaglandinsand thromboxanes. Certain ones of the preceding mediators induce thelocal generation and release of metalloproteinases, and these in turn,expose latent tissue binding sites, including intact and partiallycleaved integrins, RDGS peptides, laminin, collagen, fibronectin, andcell-surface core-protein components of glycosaminoglycans.

Cytokines, monocyte chemoattractant protein (MCP), tissuemetalloproteinases, and proteolytic tissue matrix fragments directlyinduce the local endothelium to become adhesive for circulating whiteblood cells, including neutrophils, monocytes and lymphocytes. Theinduced endothelial adhesive molecules (adhesins) include: P-selectin(gmp-140), E-selectin (ELAM-1), intercellular cell adhesion molecule(ICAM-1), vascular cell adhesion molecule (VCAM-1), inducible celladhesion molecule, (INCAM-110), von Willebrand's factor (vWF, FactorVIII antigen) (see below for disease states which activate theserespective types of endothelial adhesins). Additional cascades becomeactivated which indirectly amplify endothelial adhesiveness. Theseinclude (1) coagulation factors, especially fibronectin, tissue factor,thrombin, fibrinogen, fibrin, and their split products, especiallyfibronectin split products and fibrinopeptide A; (2) platelet-derivedfactors: platelet activating factor (PAF), glycoprotein IIb/IIIacomplex; (3) white-cell (a) L-selecting, and (b) integrins, includingVLA-4 (very late antigen 4); and (4) numerous complement factors.

The preceding pathologic processes and signals are involved, directly orindirectly as follows, in the binding and site localization of acidiccarriers, including acidic saccharides (AC) and glycosaminoglycans(GAGs) (Note that in the following outline, potential tissue bindingsites are designated as "GAGs" and "ACs").

1. Local tissue diseases induce local cytokines and mediators, asdescribed above.

2. These cytokines and mediators induce tissue chemoattractants,including MCP and IL-8, which comprise a family of arginine-rich, 8 Kd,heparin-binding proteins reported to bind GAGs/ACs Huber et al. (1991),incorporated by reference herein!;

3. The cytokines and mediators of No. 1, above, induce the localendothelium to express P-selectin, the vascular cell adhesion molecular(VCAM-1), inducible cell adhesion molecule (INCAM-110), and vonWillebrand's factor (vWF, Factor VIII antigen), which are reportedbinding determinants for GAGs/ACs Bevilaqua et al. (1993); Bevilacqua etal. (1993)!; P-selectin is reported to bind GAGs Bevilacqua et al.(1993)!;

4. Integrins, including but not limited to VLA-4, are induced oncirculating white blood cells, including lymphocytes, during variousdisease processes (see below); VLA-4 has a distinct binding site on the(induced) endothelial selectin, VCAM-1 (see No. 3, above); fibronectin,which is abundantly present in plasma and also available from tissuesites, has a distinct and separate binding site on VLA-4 Elices et al.(1990)!; since fibronectin has specific binding sites for GAGs/ACsBevilaqua et al. (1993)!, these amplification steps provide a strongadditional mechanism for site localization of GAGs/ACs;

5. The chemoattractants, MCP and IL-8, lymphocyte integrin, VLA-4,platelet factor, PAF, and coagulation factors, thrombin, fibronectin andothers, diffuse from local tissue and blood, respectively, bind to theinduced endothelial selectins, and amplify adhesiveness and activationat the initial endothelial P-selectin sites for GAGs/ACs Elices et al.(1990); Lorant et al. (1993) !;

6. Tissue metalloproteinases become activated and expose new bindingsites for GAGs/ACs in the tissues which underlie the activatedendothelia. These new tissue binding sites include as follows Ranney(1990); Ranney (1992); Travis (1993); Bevilaqua et al. (1993)!:

a. fibronectin fragments;

b. collagen fragments;

c. laminin fragments;

d. RGDS peptides;

e. Exposed core proteins of GAGs;

7. White blood cells are attracted to the site, become activated andrelease additional proteolytic enzymes, thereby amplifying No. 6 andincreasing the exposure of binding sites for GAGs/ACs in the tissuematrix.

8. GAG/AC carriers selectively bind the induced and exposed determinantslisted in Nos. 1-7, above, giving immune-type localization which isspecific for induced binding sites (lectins) at the lectin-carbohydratelevel characteristic of white-cell adhesion;

9. In cases where the carrier substance has multivalent binding to thetarget binding substance, including for example, cases in which thecarrier is an acidic oligosaccharide or polysaccharide or an acidicglycosaminoglycan, multivalent binding of the endothelial surfaceinduces rapid extravasation of the carrier and bound active, and resultsin substantially increased loading of the underlying tissue matrix,relative to that achieved by antibodies, liposomes, and monovalentbinding substances, such as hormones and monovalent-binding sugars;

10. Adhesion of GAGs/ACs to induced and exposed tissue binding sites,reduces plasma backdiffusion of GAGs/ACs and their bound actives,thereby giving sustained retention within the tissue site;

11. Controlled release of the diagnostic or drug active from carrierscomprising GAGs/ACs occurs gradually within the diseased site, therebyresulting in targeted controlled release;

12. Tumor cells, microbial targets and damaged cellular targets withinthe tissue site, may selectively take up the GAG/AC plus bounddiagnostic or drug active, based respectively, on: induced tumor aniontransport pathways, microbial binding sites for GAGs/ACs, andproteolytically exposed cell-surface core proteins Ranney 07/880,660,07/803,595 and 07/642,033!- - - Fe uptake by hepatomas, Cr4S uptakeby - - - ); Kjellen et al. (1977)!

13. In cases where the carriers are hydrophilic or essentiallycompletely hydrophilic, these carriers cause their bound actives tomigrate out (permeate) into a small rim of normal tissue around eachfocus of disease, typically comprising a rim about 30-75 um thick;however, such carriers undergo selective uptake by abnormal cells withintissue site and preferentially avoid uptake by normal cells within thesite, thereby giving:

a. In cases of diagnostic imaging applications: very sharp definition ofthe boundary between tumors or infarcts and the surrounding normaltissues;

b. In cases of therapeutic applications:

(1) protection against spread of disease at the rim;

(2) relative protection of normal cells within and adjacent to the siteof disease, from uptake of cytotoxic drugs.

14. In the case of hydrophilic carriers, including but not limited toGAGs/ACs, the non-targeted fraction of active is cleared rapidly andnon-toxically, thereby minimizing:

a. in imaging uses, background signal intensity;

b. in all uses:

(1) normal organ exposure; and

(2) systemic side effects.

Regarding the above outline, the following A. cytokines and mediators;and B. selectins, integrins and adhesins are reported to be induced byvarious disease states in addition to that reported for tumors, aboveBevilaqua et al. (1993)!. Representative non-oncologic induction alsooccurs as follows.

A. Cytokines and mediators.

1. MCP: Experimental autoimmune encephalomyelitis in mice Ransohoff etal. (1993)!;

2. IL-8: Neovascularization: Strieter et al. (1992)!;

3. PAF: Reperfused ischemic heart Montrucchio et al. (1993)!.

B. Selectins, Integrins and Adhesins.

1. ELAM-1:

a. Liver portal tract endothelia in acute and chronic inflammation andallograft rejection Steinhoff et al. (1993)!;

b. Active inflammatory processes, including acute appendicitis Rice etal. (1992)!.

2. VCAM-1:

a. Simian AIDS encephalitis Sasseville et al. (1992) !.

b. Liver and pancreas allograft rejection Bacchi et al. (1993)!.

3. INCAM-110: Chronic inflammatory diseases, including sarcoidosis Riceet al. (1991)!.

4. Integrin, beta 1 subunit cell adhesion receptor: inflammatory jointsynovium Nikkari et al. (1993)!.

It is apparent from the above, that broad categories and many specifictypes of focal tissue disease may be addressed by the carriers andactives of the present invention, both for diagnostic and therapeuticuses, including tumors, cardiovascular disease, inflammatory disease,bacterial and viral (AIDS) infections, central nervous systemdegenerative disorders, and allograft rejection. It will also be obviousto those skilled in the art, that numerous additional disease states maybe selectively addressed by the carriers disclosed in this invention.

The site selectivity of glycosaminoglycans (GAGs) appears to mimic animmune mechanism at the level of white-cell targeting rather thanantibody targeting. Because antibodies have extremely highspecificities, they characteristically miss major subregions of diseasefoci (typically as great as 60% of tumor cells are nonbinding).Recently, one of the GAG-binding determinants of endothelial P-selectinhas been identified as sialyl Lewis x. Others are in the process ofidentification. Notably, the available nonvalent oligosaccharidesspecific for sialyl Lewis x suffer from two critical problems:

1. They are exceedingly expensive materials, available only by syntheticor semi-synthetic means, and hence, are not cost effective;

2. They do not bind effectively at diseased sites under in vivoconditions, apparently due to the inability as monomeric bindingsubstances to displace endogenous interfering substances which arepre-bound at these sites.

There are two apparent benefits of the relatively broader range of GAGspecificities and redundancy of GAG binding sites present on diseasedendothelium and tissue matrix:

1. GAGs allow a broader range of tumors and diseases to be targeted thanthat possible with antibodies (which typically target only a subset ofhistologic types -- -- -- even within a given class of tumor, and hence,are typically ineffective from both a medical and cost/developmentstandpoint);

2. GAGs are projected to be effective over a greater time interval, fromearly onset of disease to progression and regression.

Despite the broader targeting specificity of GAGs over antibodies, theirfavorable clearance and avoidance of uptake by normal cells reducesystemic and local toxicities, even though more than one type of diseasesite may undergo targeted accumulation of the diagnostic/drug within itsextracellular matrix.

The polymeric and multivalent binding properties of GAGs both are veryimportant for optimal site localization of the attached diagnostic/drug.GAG molecular weights of ca. 8,000 to 45,000 MW are important in orderto:

1. Restrict initial biodistribution of the diagnostic/drug to the plasmacompartment and thereby maximize the quantity of agent available forsite targeting;

2. Displace endogenous interfering substances which are pre-bound todiseased endothelium;

3. Induce active endothelial translocation of the GAG-diagnostic/druginto the underlying tissue matrix;

4. Afford rapid clearance and markedly reduced side effects of theattached actives.

SUMMARY OF THE INVENTION

The present invention encompasses novel agents comprising cationic orchemically basic metal chelators in association with hydrophiliccarriers of anionic or chemically acidic saccharides, sulfatoids andglycosaminoglycans. In certain embodiments of the invention, the agentsalso comprise chelated metals and metal ions. The binding of the metalchelators to the carriers is stabilized by covalent or non-covalentchemical and physical means. In some embodiments, novel non-covalentlybound compositions give uniquely high payloads and ratio of metalchelator to carrier, ranging from a low of about 15% metal chelator byweight, to a characteristic range of 70% to 90% metal chelator byweight. Specific embodiments comprise deferoxamine, ferrioxamine,iron-basic porphine, iron-triethylenetetraamine, gadolinium DTPA-lysine,gadolinium DOTA-lysine and gadolinium with basic derivatives ofporphyrins, porphines, expanded porphyrins, Texaphyrins and sapphyrinsas the basic or cationic metal chelators, which are in turn, bound toacidic or anionic carriers, including one or more of acidic or anionicsaccharides, and including sulfated sucrose, pentosan polysulfate,dermatan sulfate, oversulfated dermatan sulfate, chondroitin sulfate,oversulfated chondroitin sulfate, heparan sulfate, beef heparin, porcineheparin, non-anticoagulant heparins, and other native and modifiedacidic saccharides and glycosaminoglycans.

Methods of magnetic resonance image (MRI) contrast enhancement are aparticular embodiment of the present invention which confirm very rapid,carrier-mediated, site-selective in vivo localization and sustained siteretention of metal-chelator compositions, based on stable binding of themetal chelator and carrier during in vivo plasma transit, allowing sitelocalization following intravenous administration. Rapid and selectiveendothelial-site binding, facilitated rapid extravasation intounderlying tissue sites, site accumulation, sustained site retention,together with rapid clearance of the non-site-localized fraction arealso demonstrated by the use of the compositions of the presentinvention in the selective MRI contrast enhancement of tumors andcardiovascular infarcts.

Surprising and unexpected improvements of selectivity, mechanism oflocalization and cellular uptake, and MRI contrast sensitivity are shownfor metal chelates having standard paramagnetic potencies. Furtheradvantages of the use of the compositions and methods of the presentinvention are delineated in the examples (infra) including specialhistologic staining evidence which confirms the site-selectiveendothelial binding, extravasation, tissue matrix accumulation andcellular uptake mechanism. Selective localization and MRI imagingefficacy are also shown to occur when paramagnetic metal chelatoractives are administered in carrier-bound form but not in free form.

In its most general embodiment, the present invention is an agentcomprising a chelator for metal ions, said chelator having a cationicgroup and being bound to an anionic, hydrophilic carrier. In alternateembodiments, the chelator for metal ions which has a cationic group isbound to an anionic, hydrophilic carrier by non-covalent electrostaticbinding. And, in certain alternate embodiments the invention comprisesan agent comprising a basic chelator for metal ions, said chelatorhaving a cationic group and being covalently bound to an anionic,hydrophilic carrier. In the particular embodiments of the invention inwhich the chelator is not covalently bound to the carrier, the saidchelator may be basic.

In certain embodiments of the present invention, the agent whichcomprises a chelator for metal ions and having a cationic group bound toan anionic hydrophilic carrier may further comprise a chelated metalion, and in particular it may further comprise a paramagnetic metal ion.The agents of the present invention, in particular those which comprisethe chelator for metal ions non-covalently bound to the carrier may befurther defined as being at least about 15 weight percent chelator.Preferably, the chelator has a formation constant for paramagnetic metalions of at least about 10¹⁴.

Those agents of the present invention which comprise a metal ion willpreferably comprise a metal ion selected from the group consisting ofiron, manganese, chromium, copper, nickel, gadolinium, erbium, europium,dysprosium and holmium. In certain embodiments, the agents of thepresent invention may even comprise a metal ion selected from the groupconsisting of boron, magnesium, aluminum, gallium, germanium, zinc,cobalt, calcium, rubidium, yttrium, technetium, ruthenium, rhenium,indium, iridium, platinum, thallium and samarium. It is understood thatother metal ions which are functionally equivalent to the listed metalions are also included and would fall within the scope and spirit of thepresently claimed invention.

In certain preferred embodiments of the invention, the agents comprise acarrier wherein said carrier is an acidic saccharide, oligosaccharide,polysaccharide or glycosaminoglycan. The carrier may also be an acidicglycosaminoglycan or sulfatoid. In particular, the carrier may be, butis not limited to heparin, desulfated heparin, glycine-conjugatedheparin, heparan sulfate, dermatan sulfate, chondroitin sulfate,hyaluronic acid, pentosan polysulfate, dextran sulfate, sulfatedcyclodextrin or sulfated sucrose.

In certain embodiments of the invention, the chelator is a chelator ofiron ions. Preferably the chelator is a hydroxamate, and more preferablyit is deferoxamine. In certain preferred embodiments the chelatortogether with the metal ion is ferrichrome, ferrioxamine, enterobactin,ferrimycobactin or ferrichrysin. In a particularly preferred embodiment,the chelator is deferoxamine, the carrier is heparin, or a heparinfragment and the agent further comprises iron(III). In an alternateembodiment, the chelator is deferoxamine and the carrier is dermatansulfate or a dermatan sulfate fragment and the agent may furthercomprise chelated iron(III).

In a certain embodiment, the invention may also comprise deferoxaminebound to a carrier selected from the group consisting of heparin,heparan sulfate, dermatan sulfate or chondroitin sulfate, and mayfurther comprise a metal ion. The agents of the present invention mayalso comprise a chelator which is a porphine, porphyrin, sapphyrin ortexaphyrin and which may further comprise a metal ion, and preferably aniron ion or a gadolinium ion.

In a particularly preferred embodiment the agent of the presentinvention may comprise a chelator which is5,10,15,20-Tetrakis(1-methyl-4-pyridyl)-21H,23-porphine, a carrier whichis heparin and a chelated iron ion. In certain embodiments, the chelatormay also be a polyaminocarboxylate or macrocyclic, and preferably abasic or amine derivative of diethylenetriaminetetraacetate, or morepreferably a basic or amine derivative of1,4,7,10-tetraazacyclododecane-N,N',N","'-tetraacetate (DOTA). In theagents of the present invention, the carrier may also be defined furtheras being complementary to endothelial determinants selectively inducedat disease sites.

In a certain embodiment, the present invention is an image-enhancingagent or spectral-enhancing agent to enhance images arising from inducedmagnetic resonance signals, the agent comprising ferrioxamine covalentlyconjugated to heparin by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide,N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, or carbonyldiimidazole.Alternatively, the invention is a spectral-enhancing agent to enhanceimages arising from induced magnetic resonance signals, the agentcomprising Gd(III)diethylenetriaminepentaacetate covalently conjugatedto one of heparin, dermatan sulfate or chondroitin sulfate. In anotheralternative, the invention is an agent for in vivo imaging, the agentcomprising a basic chelator for metal ions and chelated metal ion, saidchelator being bound by non-covalent electrostatic binding to ahydrophilic carrier selected from the group consisting of heparin,desulfated heparin, glycine-conjugated heparin, heparan sulfate,dermatan sulfate, chondroitin sulfate, hyaluronic acid, pentosanpolysulfate, dextran sulfate, sulfated cyclodextrin or sulfated sucrose.The agent for enhancing body imaging preferably comprises deferoxamine,chelated Fe(III) and a glycosaminoglycan carrier bound to saiddeferoxamine and more preferably the glycosaminoglycan carrier isdermatan sulfate, and/or the Fe(III) is a radiopharmaceutical metal ion,and most preferably the radiopharmaceutical metal ion is ⁵⁹ iron or ⁶⁷gallium.

In an alternate embodiment, the invention is an agent for enhancing bodyimaging, the agent comprising diethylenetriaminepentaacetate-lysine,chelated Gd(III) and a glycosaminoglycan carrier bound to saiddiethylenetriaminepentaacetate-lysine. Alternatively, the invention isan agent for enhancing body imaging, the agent comprising DOTA-lysine,chelated Gd(III) and a glycosaminoglycan carrier bound to said1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetate-lysine(DOTA-lysine). In a particular embodiment, the invention is an agentcomprising ferrioxamine bound by non-covalent electrostatic binding todermatan sulfate.

It is understood that any of the agents of the present invention asdescribed in the above paragraphs or in the appended claims may bedefined further as being in a combination with at least one of a buffer,saccharide, sulfated saccharide, or salt, to produce an osmotic strengthsuitable for parenteral administration, and as being an aqueous solutionor a lyophilized or dry preparation suitable for aqueous reconstitutionhaving the desired osmotic strength, and wherein said agent is asepticor sterile.

Another embodiment of the invention is a method of enhancing magneticresonance images or spectra in vertebrate animals comprisingadministering to said animal an effective amount of an agent of theinvention which comprises the metal ion chelator, the carrier asdescribed and a paramagnetic ion. In particular, the invention is amethod of enhancing in vivo images arising from induced magneticresonance signals, comprising the steps of administering to a subject aneffective amount of an agent of the present invention which comprises aparamagnetic ion, exposing the subject to a magnetic field andradiofrequency pulse and acquiring an induced magnetic resonance signalto obtain a contrast effect.

In an alternative embodiment, the invention is a method of enhancing invivo images, comprising the steps of administering to a subject aneffective amount of an agent of the present invention which comprises achelated metal ion, exposing the body to ultrasound or X-rays andmeasuring signal modulation to obtain a contrast effect.

In another embodiment, the invention is a method of obtaining in vivobody images comprising administering to a subject an effective amount ofan agent of the invention which comprises a metal ion wherein the metalion is a radioisotope and measuring scintigraphic signals to obtain animage.

In another embodiment, the invention is a method of treating vasculardisease, comprising administering to a subject a therapeuticallyeffective amount of an agent of the present invention, and preferably anagent which comprises a metal ion.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings and figures are presented to illustrate preferredembodiments of the present invention and their uses in MRI contrastenhancement. These examples are purely illustrative, and do not in anyway delimit the full scope of the present invention.

FIG. 1A is a control infrared spectrum of diethylenetriaminetetraacetate(DTPA) substrate (see Example 3).

FIG. 1B is a control infrared spectrum of L-lysine.HCl substrate (seeExample 3).

FIG. 1C is a control infrared spectrum of a physical mixture of theseDTPA and L-lysine.HCl substrates without any chemical covalent linkageof the two substrates (see Example 3).

FIG. 1D is the experimental infrared spectrum of L-lysine covalentlyconjugated to DTPA by 1-ethyl-3-(3dimethylaminopropyl) carbodiimide(EDC) linkage (see Example 3). Note the changes (height, width and lossof splitting) in signature peaks in the range of 1250-1700 wavenumbers,which indicate covalent conjugate formation.

FIGS. 2A-4D and 8A-C show T1-weighted MRI images (TR/TE=800/45, 550/23and 600/45) performed at 1.0 and 1.5 Tesla, before (Pre) and after(Post) intravenous (i.v.) injection of Ferrioxamine:Dermatan SulfateSelective Paramagnetic Contrast Agent, prepared as in Examples 2 and 5,and injected i.v. at a Ferrioxamine dose of 0.155 mmol/Kg into Fisher344 female rats, with syngeneic breast adenocarcinoma inoculatedpreviously into the liver, such that tumor diameters at the time ofimaging are between 1.0 cm and 2.5 cm.

FIG. 2A Precontrast image of liver (tumor not conspicuous).

FIG. 2B Liver image at 7 min postinjection (MPI) of the SelectiveParamagnetic Contrast Agent, Ferrioxamine:Dermatan Sulfate (0.155mmol/Kg) i.v., showing marked contrast enhancement of tumor in rightlobe of liver, very sharp tumor boundaries against surrounding liver,and discretely demarcated darker central region of tumornecrosis--allowing tumor perfusion and function to be spatially resolvedand assessed within different, very small anatomical subregions.

FIG. 3A Precontrast image of liver (tumor is present but notconspicuous).

FIG. 3B Liver image at 7 MPI of Ferrioxamine Active Alone (without anyDermatan Sulfate Carrier). Note that acute contrast enhancement is onlyvery slight or nonexistent. This differs markedly from the pronouncedtumor enhancement seen in FIG. 2B; and it indicates that binding of theFerrioxamine active by the Dermatan Sulfate carrier is a requirement fortumor-site localization and tumor uptake of Ferrioxamine active.

FIG. 4A Precontrast T1 image (TR/TE =800/45) of liver (breast tumor ispresent but not conspicuous).

FIG. 4B Liver image at 21 MPI of Ferrioxamine:Dermatan Sulfate SelectiveMRI Contrast Agent. Note the marked enhancement of main tumor mass anddistinct tumor borders. Also note the small, 2-mm, bright enhancement oftumor metastasis in left lobe of liver. This metastasis is completelynon-visualized in the Precontrast T1 images.

FIG. 4C Liver image at 30 MPI of Ferrioxamine:Dermatan Sulfate SelectiveMRI Contrast Agent. Note the sustained enhancement of main tumor andmetastasis.

FIG. 4D Liver image at 42 MPI of Ferrioxamine:Dermatan Sulfate SelectiveMRI Contrast Agent. Note: continued strong enhancement of main tumor andmetastasis at prolonged postcontrast interval, at high, sustainedsensitivity, and with continued delineation of tumor boundaries in bothnodules (selectivity), plus delineation of the very small non-perfusedregion centrally within the 2-mm liver metastasis.

FIG. 5 Region-of-interest (ROI) analyses of MRI image intensities from atumor animal analogous to that shown in FIGS. 4A-D. Upper line=tumorROI's; Lower line=liver ROI's; time points=Precontrast; and 12, 27, 44and 64 MPI of Ferrioxamine:Dermatan Sulfate Selective MRI ContrastAgent. Note the Intensity Ratios of Tumor to Liver are: (a) at the Peaktime of 12 MPI=11:1; (b) as an average over the 27-64 MPI=3.2:1--both(a) and (b) additionally indicating very good selectivity for tumorversus liver. Intensity fades only very gradually with time, unlike thekinetics reported for Gd:DTPA, which are very rapid and have a t1/2 atthe site of ca. 12-20 min (images not shown).

FIG. 6 Special histologic stain (heated ferroferricyanide reaction) offormalin-fixed section of Outer Tumor Rim 7-10 MPI ofFerrioxamine:Dermatan Sulfate Selective MRI Contrast Agent. Noteselective staining for ferrioxamine iron (a) strongly positive on andwithin tumor endothelium, (b) strongly positive in the subendothelia,(c) moderately positive in the extracellular matrix of tumor, and (d)lightly to moderately positive within tumor intracellular sites.

FIG. 7A Same stain, conditions, and post-contrast time as FIG. 6, excepttissue section is taken from Central Tumor, 7-10 MPI ofFerrioxamine:Dermatan Sulfate Selective MRI Contrast Agent. Significantstaining positivity is present at all sites as in FIG. 6.

FIG. 7B Identical to 7A, except 7-10 MPI of Ferrioxamine Active Alone.Note the complete absence of staining positivity. This correlatesdirectly with the results of MRI imaging with the full Agent (Activebound to Carrier) versus that with Active Alone (Active in freeform)--(refer to FIGS. 2A and B versus 3).

FIG. 8A T1-weighted (TR/TE=600/45) image of Lung Field in rat withprimary liver breast tumor. Note that the lung metastases (2-mm to 3-mmnodules) are only faintly conspicuous Precontrast.

FIG. 8B Lung Field of same rat at 12 MPI. Note the marked improvement insensitivity of tumor detection (conspicuity) due to selective, brightenhancement of the lung metastases. Also note the sharpness of tumorboundaries.

FIG. 8C Same Lung Field at 17 MPI--showing sustained enhancement andsustained sharpness of tumor boundaries. By comparison, the rapiddiffusion rates of Gd:DTPA lead to rapidly fuzzy boundaries at earlytimes; and thereby also decrease the sensitivity of detecting pulmonarymetastases.

FIGS. 9A-E and 10A-E show T1-weighted MRI images (TR/TE=250/80)performed at 4.7 Tesla, before (Pre) and after (Post) intravenous (i.v.)injection of Ferrioxamine:Dermatan Sulfate Selective ParamagneticContrast Agent (FIGS. 9A-E) prepared as in Examples 2 and 5, andinjected i.v. at an Iron(III) dose of 0.155 mmol/Kg; compared toGadolinium DTPA dimeglumine (FIGS. 10A-E), injected i.v. at a Gd(III)dose of 0.100 mmol/Kg; each of these agents being administered toCopenhagen rats with syngeneic AT-1 prostate adenocarcinoma inoculatedinto previously prepared skin pouches Hahn et al. (1993)!, such thattumor diameters at the time of imaging are between 1.0 cm and 2.5 cm.

FIG. 9A Precontrast image for Ferrioxamine:Dermatan Sulfate SelectiveContrast Agent.

FIG. 9B 7 MPI of Ferrioxamine:Dermatan Sulfate, liquid form at aferrioxamine concentration of 0.166 mmol/mL. Note the strong enhancementof Outer Rim and Vascular array which fans out from the tumor pedicle.

FIG. 9C Same as 9B, except 20 MPI. Note the sustained, discreteenhancement of elements in FIG. 9B

FIG. 9D Same as 9C, except 40 MPI. Note the sustained contrast anddelineation of Outer Rim.

FIG. 9E Same as 9D, except 60 MPI. Note the onset of contrast fading.

FIG. 10A Precontrast image for Gd:DTPA dimeglumine Nonselective ContrastAgent.

FIG. 10B 7 MPI of Gd:DTPA dimeglumine. Note that the Outer Rim is notwell delineated, even at this very early post-contrast interval.

FIG. 10C Same as 10B, except 20 MPI. Note the marked early contrastfading overall, with some agent sequestration seen at the central,poorly perfused (cystic) regions of tumor (as is typically reported forGd:DTPA when used for imaging at body sites).

FIG. 10D Same as 10C, except 40 MPI. Note that enhancement is nearlyreverted to background levels.

FIG. 10E Same as 10D, except 60 MPI. No residual contrast, except forcentral cystic regions.

FIGS. 11A-D show T1-weighted MRI ECG-gated cardiovascular imagesperformed at 0.5 Tesla, before (Pre) and after (Post) rapid intravenous(i.v.) infusion of Ferrioxamine:Dermatan Sulfate Selective ParamagneticContrast Agent prepared as in Examples 2 and 5, and injected i.v. at anIron(III) dose of 0.155 mmol/Kg into German Shepherd dogs with acute,90-min myocardial infarcts (ligature of proximal left anteriordescending coronary artery) followed by reperfusion for ca. 90 minutesprior to contrast agent infusion.

FIG. 11A Precontrast image.

FIG. 11B 7 MPI, showing strong enhancement of infarct byFerrioxamine:Dermatan Sulfate Agent, and in particular delineating theboundary of the infarct--putatively the boundary of the marginal zone.Note the central darker region--putatively the irreversible centralinfarct zone.

FIG. 11C 20 MPI, showing sustained strong enhancement and zones asabove.

FIG. 11D 40 MPI, same as 11C, except filling in of central zone; absenceof significant overall contrast fading. NOTES: (1) injection ofFerrioxamine Agent Alone at 0.155 mmol/Kg, gives no detectableenhancement (images not shown); (2) infarct sizes and positions aredocumented by double dye infusion methods immediately after imaging.

FIGS. 12A-D show MRI 4.7 Tesla, T1-weighted images of Copenhagen ratswith the AT-1 prostate tumor model (as in FIGS. 9A-E and 10A-E), butrats are injected i.v. with Ferrioxamine:Dermatan Sulfate SelectiveContrast Agent in the lyophilized (versus liquid) form, and the Agent isreconstituted with water just prior to administration at a higherconcentration of 0.415 mmol/mL Fe(III) and administered at the usualdose of 0.155 mmol of Fe(III) per Kg.

FIG. 12A Precontrast image for Ferrioxamine:Dermatan Sulfate SelectiveContrast Agent.

FIG. 12B 7 MPI of Ferrioxamine:Dermatan Sulfate, lyophilizedreconstituted to a Fe(III) concentration of 0.415 mmol/mL. Note the verystrong enhancement of the entire Outer Rim of tumor.

FIG. 12C Same as 12B, except 20 MPI. Note the sustained, very strongenhancement and delineation of Outer Rim.

FIG. 12D Same as 12C, except 40 MPI. Note the sustained very strongenhancement of Outer Rim with the Central Tumor now also starting toenhance brightly. Also note there is virtually no contrast fading at 40minutes.

FIGS. 13A-D show MRI 4.7 Tesla, T1-weighted images of Copenhagen ratswith the AT-1 prostate tumor model (as in FIGS. 12A-D), but rats areinjected i.v. with Gd(III):DTPA-Lys:Dermatan Sulfate Selective ContrastAgent in liquid form pre-concentrated to 0.415 mmol/mL Gd(III) andadministered at the usual dose of 0.155 mmol of Gd(III) per Kg.

FIG. 13A Precontrast image for Gd(III):DTPA-Lys:Dermatan SulfateSelective Contrast Agent.

FIG. 13B 7 MPI of Gd(III):DTPA-Lys:Dermatan Sulfate, at 0.415 mmol/mL.Note the exceedingly strong enhancement of the entire Outer Rim as wellas Central Tumor. This is consistent with the higher paramagneticpotency of Gd:DTPA chelate, R1=4.3 mmol.sec!-1, relative to ferrioxaminechelate, R1=1.5-1.8 mmol.sec!-1.

FIG. 13C Same as 13B, except 20 MPI. Note the sustained, very strongabsolute enhancement Outer Rim. Also note additionally strongenhancement of the central vascular array (as differentiated from cysticsequestration).

FIG. 13D Same as 13C, except 40 MPI. Note sustained enhancement of OuterRim, with overall enhancement just beginning to fade at 40 minutes, butabsolute enhancement remaining as bright or brighter in all regionsrelative to Ferrioxamine:Dermatan Sulfate.

FIGS. 14A-D show MRI 4.7 Tesla, T1-weighted images of Copenhagen ratswith the AT-1 prostate tumor model (as in FIGS. 13A-D), but rats areinjected i.v. with Ferrioxamine Selective Contrast Agent, wherein theActive is non-covalently bound to Oversulfated Dermatan Sulfate, theAgent lyophilized and reconstituted with water just prior toadministration at a concentration of 0.332 mmol/mL Fe(III) andadministered at the usual dose of 0.155 mmol of Fe(III) per Kg.

FIG. 14A Precontrast.

FIG. 14B 7 MPI.

FIG. 14C 20 MPI.

FIG. 14D 40 MPI. Note the slightly greater enhancement of Tumor Rim andgreater definition of the vascular array at all times, in relation toFerrioxamine bound to Native Dermatan Sulfate (above)

FIGS. 15A-D show MRI 4.7 Tesla, T1-weighted images of Copenhagen ratswith the AT-1 prostate tumor model (as in FIGS. 13A-D), but rats areinjected i.v. with Ferrioxamine Selective Contrast Agent, wherein theActive is non-covalently bound to Oversulfated Chondroitin Sulfate, theAgent lyophilized and reconstituted with water just prior toadministration at a concentration of 0.332 mmol/mL Fe(III) andadministered at the usual dose of 0.155 mmol of Fe(III) per Kg.

FIG. 15A Precontrast.

FIG. 15B 7 MPI.

FIG. 15C 20 MPI.

FIG. 15D 40 MPI. Note the moderately greater enhancement of Tumor Rimand greater definition of the vascular array at all times, in relationFerrioxamine bound to Native Dermatan Sulfate (above).

FIGS. 16A-D show MRI 4.7 Tesla, T1-weighted images of Copenhagen ratswith the AT-1 prostate tumor model (as in FIGS. 13A-D), but rats areinjected i.v. with Ferrioxamine Selective Contrast Agent, wherein theActive is non-covalently bound to a non-anticoagulant GAG, HeparanSulfate, the Agent lyophilized and reconstituted with water just priorto administration at a concentration of 0.332 mmol/mL Fe(III) andadministered at the usual dose of 0.155 mmol of Fe(III) per Kg.

FIG. 16A Precontrast.

FIG. 16B 7 MPI.

FIG. 16C 20 MPI.

FIG. 16D 40 MPI. Note the very homogeneous enhancement of Outer Rim andCentral Tumor at virtually all post-contrast times, in relation to thedifferential Rim enhancement achieved by essentially all of the otherGAG carriers. This property may be useful in certain therapeuticapplications.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The many innovative teachings of the present invention will be describedwith particular reference to the presently preferred embodiments,wherein these innovative teachings are advantageously applied to theparticular issues of in vivo T1-Type MRI image contrast enhancement bysite-selective localization and sustained site retention of paramagneticmetal chelates according to optimal spatial and kinetic profiles at thesite, while simultaneously enhancing clearance and minimizing toxicityof the non-localized dose fraction. However, it should be understoodthat this principal embodiment is only one example of the manyadvantageous uses of the innovative teachings herein. For example, thevarious types of innovative compositions and methods disclosed hereincan alternatively be used to selectively localize and enhance clearanceof radionuclide imaging agents, X-ray contrast agents,ultrasound-acoustic image enhancing agents and a wide spectrum oftherapeutic agents which are based on site delivery of metal chelatesand in situ chelation of endogenous body metals. Of special interest tothe therapeutic agents and uses embodied herein, are actives andindications useful in oncotherapy, cardiovascular infarcts, restenosis,atherosclerosis, acute thrombosis, microvascular disease, inflammation,and any other tissue diseases which have as part of their development orprogression, a vascular component amenable to modulation by the novelteachings, compositions and uses described herein. Hence, it will beobvious to those skilled in the art, that numerous additionalcompositions and uses are uniquely enabled by the present invention. Thefollowing examples are presented to illustrate preferred embodiments ofthe present invention, their uses in MRI contrast enhancement. Theseexamples are purely illustrative, and do not in any way delimit the fullscope of the present invention.

The present invention specifically describes the preparation andutilization of novel contrast agents for magnetic resonance imaging.These novel contrast agents consist of paramagnetic metal chelates, asdistinguished from metal-atom complexes, wherein the presently disclosedchelates are bound to glycosaminoglycans (GAG). Binding of the metalcomplex to the GAG may take the form of, but is not limited to,electrostatic interactions (ion-paired), hydrogen-bonding, Van der Waalsinteractions, covalent linkages, or any combination of theseinteractions. Following parenteral administration of these metalcomplex-glycosaminoglycan formulations, the technology described hereinutilizes a biocompatible carrier molecule to deliver an associatedbiologically active substance to sites of vascular injury.

The present invention provides substantially improved MRI image andspectral enhancement compositions and methods, whereby the capacity ofMRI hardware systems to detect tumors, cardiovascular diseases, andother diseases with a vascular or endothelial adhesive component aregreatly enhanced. These improvements are presently accomplished byintroducing a chelated paramagnetic metal ion selectively into tissuesites of interest, inducing selective (local) modulation of T1-Type,paramagnetic relaxation of water protons or other diffusible nucleipresent within the site which are susceptible to orientation by fixedand gradient magnetic fields and to pulsed re-orientation byradiofrequency fields of appropriate resonant frequencies, therebygiving rise to detectable modulations of induced magnetic resonancesignals, in the forms of either image contrast enhancement or spectralenhancement.

Past disclosures (Ranney: U.S. Ser. No. 07/880,660, filed May 8, 1992,U.S. Ser. No. 07/863,595 filed Apr. 3, 1992, now U.S. Pat. No. 5,214,661and U.S. Ser. No.07/642,033 filed Jan. 1, 1991! have dealt with thebinding of magnetic agents which required: (a) magnetic potenciesgreater than that of the most potent single metal ion, gadolinium(III);(b) intramolecularly coupled, polyatomic metal-atom complexes stabilizedby non-bridged ligands which have a stronger potential for chemical andphysical instability than the stably, bridged-ligand chelated metal ionsdisclosed herein; and (c) divalent cationic charge on the"superparamagnetic" active for binding to anionic carriers, versus thepresently disclosed requirement for only a monovalent cationic charge onparamagnetic metal chelator actives. It is understood, that for MRIuses, the metal chelator will also comprise an appropriate paramagneticmetal ion, for example, preferably iron(III) or gadolinium (III),however, for certain other diagnostic and therapeutic compositions anduses, the presently disclosed metal chelators may either comprise oravoid an appropriate metal ion. For the presently preferred MRIapplications, basic metal chelators and metal chelators withelectrophilic properties at formulation pH's are preferred, for example,ferrioxamine Crumbliss, 1991!, basic or amine derivatives of thepolyaminocarboxylate chelator, diethylenetriaminepentaacetate (DTPA),and basic or amine derivatives of the macrocyclic chelator,1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetate (DOTA) Li et al.1993; Brechbiel et al. 1986!. In certain instances and with certainpotent carriers bound to these and related actives, site localizationmay be so pronounced that the inherent potency (in vitro paramagneticR1) of the paramagnetic metal ion may not be crucial to obtainingoptimal site-localized image contrast or spectral enhancement effects.Hence, the present invention discloses pronounced T1 image contrasteffects for the basic metal chelate, ferrioxamine, which by virtue ofchelated Fe(III) ions, has a potency, or R1 relaxivity, of about 1.6-1.8mmol.sec!-1. Alternatively, basic metal chelates of Gd(III) maybeexpected under certain but not all in vivo conditions, to have apotentially greater relaxivity, due to its greater in vitro R1 of about4.0-4.3 mmol.sec!-1 when chelated by DTPA, and potentially moderatelyhigher when chelated by DOTA Geraldes et al. 1985!. Alternative metalions may preferably include the divalent or trivalent cations,manganese, chromium and dysprosium; and less preferably, those ions ofcopper, nickel, erbium, europium, and holmium.

Preferred chelators of the present invention include those with aformation constant of at least about 10¹⁴ for strongly paramagneticmetal ions disclosed above, and including a basic or cationic group.These chelators preferably include ferrioxamine, basic or aminederivatives of DOTA, DTPA, porphines, porphyrins, sapphyrins ortexaphyrins, which can preferably chelate Fe(III) and most preferablychelate Gd(III), as well as bind by principally paired-ion(electrostatic) means to the acidic groups of acidic carriers. Forexample, certain texaphyrins have an expanded macrocyclic ring whichmay, in certain instances, stably chelate Gd(III) Sessler et al. '065;Sessler et al. '720; Sessler et al. '498, incorporated by referenceherein!. Whereas texaphyrins and sapphyrins are not exemplified in thepresent invention, it will be obvious to those skilled in the art, fromthe references cited just above, and from the presently disclosed andexemplified Fe(III) chelator,5,10,15,20-Tetrakis(1-methyl-4-pyridyl)-21-23-porphine, that the relatedtexaphyrins and sapphyrins and their basic, cationic and aminederivatives, as well as the presently disclosed porphine derivative andits analogues and basic, cationic and amine derivatives, would beincluded under the disclosures and teachings of the present invention,and may be used in combination with the presently disclosed acidiccarriers. There are hybrid considerations of, among others: (a)paramagnetic potency of the metal chelate; (b) binding stability to theacidic carrier; and (c) formulation compatibility; and (d)biocompatibility and clearance in vivo. Hydrophilic chelators andcarriers are usually, but not always preferred, due to their typicallyfavorable formulation properties (absence of aggregation),biodistribution properties (absence of generalized binding tohydrophobic plasma and cell-membrane constituents during the process oflocalization); and clearance plus toxicity advantages. Alternativechelators may include the hydroxamates, ferrichrome, enterobactin,ferrimycobactin, ferrichrysin, and their basic or amine derivatives, allderivatives being defined as subsumed under the parent chelators listedabove.

Preferred carriers include monomeric, oligomeric and polymericsubstances which contain or comprise anionic or acidic groups defined atthe pH's used for formulation. These typically contain or comprisegroups of carboxylate, and more preferably, the even more stronglyacidic groups of phosphate, and most preferably, sulfate. Preferredcarriers include, but are not limited to an acidic saccharide,oligosaccharide, polysaccharide, glycosaminoglycan or sulfatoid,typically of bacterial or semi-synthetic origin, or derivatives ormodifications or fragments of the preceding substances, all definedherein as being subsumed under the names of the parent substances andcategories. Hence, preferred carriers include the following: heparin,desulfated heparin, glycine-conjugated heparin, heparin sulfate,dermatan sulfate, chondroitin sulfate, pentosan polysulfate, andsulfated sucrose, including sucrose octasulfate, and any derivative,modification or modified form thereof. Less preferably for typical MRIformulations and uses, are include the carriers of sulfatedcyclodextrin, dextran sulfate and hyaluronic acid, although any of thesemay be particularly suitable for certain specific diagnostic ortherapeutic formulations and uses.

In all cases reported and tested, non-covalent binding of the basicamine chelator to the acidic carrier gives payloads of active agentwhich are markedly higher than those afforded by covalent conjugation.For example, and preferably, ferrioxamine and Gd(III) DTPA-lysine arebound to their acidic glycosaminoglycan carriers at weight ratios of≧70%. Alternative covalent active-carrier conjugates may be preferred incertain instances, and preferred examples thereof are shown for MRIapplications.

Specific embodiments of the present invention which have been tested invivo, include, but are not limited to the presently exemplifiedpreferred embodiments of: (a) deferoxamine, (b) ferrioxamine and (c)Gd(III):DTPA-lysine basic metal chelates bound by most preferablynon-covalent means, and also preferably by covalent means, asexemplified below, to acidic glycosaminoglycans, including preferably,dermatan sulfate, chondroitin sulfate, heparan sulfate, and heparin,which include by definition, any derivative or modification thereof,including oversulfation and modification undertaken to reduceanticoagulant activities or provide improved site binding, enhancedclearance or other desired formulation or in vivo properties.Alternative preferred Agents obvious from the present disclosures, tothose skilled in the art, may induce arginine and histidine basicderivatives of DTPA and DOTA, and also of the various texaphyrins,sapphyrins, porphines, porphyrins, EHPG, and by definition, mostpreferably for MRI applications, comprising their Gd(III) and Fe(III)metal-ions, an also preferably comprising their alternative paramagneticmetal ion chelates, as disclosed above.

The present invention describes the preparation and utilization of anovel MRI contrast agent for enhancement of solid tumors andcardiovascular infarcts. The contrast agents consist of cationic orbasic paramagnetic metal complexes in association with strongly acidic,including polysulfated carriers, and including preferablyglycosaminoglycans. It would be obvious to those skilled in the art thatany acidic glycosaminoglycan can be used. Of the paired-ion systemsdescribed below, most notably are those consisting of ferrioxamine withglycosaminoglycans and DTPA-lysine with glycosaminoglycans.

It is envisioned that alternative diagnostic and therapeuticcompositions and applications may be carried out using compositionssubstantially similar to those disclosed above. For example, alternativemetal ions may be chelated for purposes of metal-ion exchange at thesite. Hence, the present formulations may contain or comprise metal ionsof manganese, aluminum, germanium, zinc, cobalt, calcium, platinum, orothers. Alternatively, for purposes of radiation and radionuclidetherapy, such compositions may contain or comprise metal ions of boron,cobalt, rubidium, yttrium, technetium, ruthenium, rhenium, indium,iridium, thallium, samarium or others. Specifically, and in some casespreferably, ⁵⁹ Fe and ⁶⁷ Ga Hashimoto et al. 1983; Janoki et al. 1983!may be substituted as radionuclide forms of the non-radioactive metalions, for purposes of nuclear medical imaging of tumors, thrombi, andother biomedical imaging purposes.

The preceding discussion is presented to specify major aspects of theinvention and their use in in vivo diagnostic and therapeuticapplications, however, to those skilled in the art many additional andrelated compositions and methods of use will be obvious from thispreceding discussion and are encompassed by the present invention.

                  TABLE 1                                                         ______________________________________                                        Advantages of Metal Ion Chelator and                                          Anionic, Hydrophilic Carrier                                                           Selective                                                            Technology                                                                             MRI Agent Antibodies                                                                             PEG      Liposomes                                ______________________________________                                        Property                                                                      Drug Payload                                                                           High 77.5%                                                                              Very Low Low 10-30%                                                                             Low                                                         5%                15-20%                                   Localization                                                                           Yes       Very Low No       No                                       In Tissue                                                                     Sites                                                                         Selectivity                                                                            Broad     Narrow   None     None                                              Immune    Immune                                                              (CHO-     (Ab-                                                                lectin)   antigen)                                                   Time to  Very Rapid                                                                              Slow     Slow     Very Slow                                Target   (several  (several (many hrs)                                                                             (hrs-days)                                        mins)     hrs)                                                       Time to  Rapid     Very Slow                                                                              Very Slow                                                                              Extremely                                Clear Plasma                         Slow (RES)                               & Body                                                                        Applications                                                                           Broad     Narrow   Narrow   Narrow                                            (Tissue   (Intravasc                                                                             (Enzymes)                                                                              (RES)                                             Sites)    ular)                                                      ______________________________________                                    

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Preparation of Deferoxamine Free Base and Use in Formulationof Ferrioxamine

The free base of deferoxamine is used in certain instances, in order tominimize the residual salt content present in final formulations whichutilize deferoxamine as a basic metal chelator. In these instances,deferoxamine is precipitated out of aqueous salt solutions by theaddition of 2 N KHCO₃, as previously reported Ramirez et al. (1973),incorporated by reference herein!. A saturated solution of deferoxamine(320 mg/mL at 25° C.) is prepared by dissolving 4.0 g of deferoxaminemesylate salt in 12.5 mL of pharmaceutical-grade water. The solution iscooled to 4° C. in an ice bath and 2.5 mL of 2.0N KHCO₃ added. The glasscontainer is scratched with a stainless steel spatula to initiateprecipitation. The precipitate is collected by centrifugation, washedrepeatedly with ice cold water, and filtered. The crude deferoxaminefree base is purified by sequential recrystallization from hot methanol.The resulting pure deferoxamine free base is dried under a stream ofnitrogen. The infrared spectrum of the deferoxamine as prepared isconsistent with that referenced above.

Ferrioxamine is formulated from the deferoxamine free base by additionof ferric chloride at stoichiometric molar ratios of Fe(III) todeferoxamine free base. This results in chelated iron and minimizesresidual mesylate and chloride ions.

EXAMPLE 2 Preparation of Ferrioxamine-Iron (III) Chelate

Batch quantities of the Fe(III) chelate of deferoxamine are prepared asfollows. Deferoxamine mesylate (methanesulfonate) (Ciba-Geigy Limited,Basel, Switzerland), 390 g, is dissolved in pharmaceutical-grade water.Alternatively, the chloride salt of deferoxamine may be used. A highlypurified slurry of ferric iron in the form of Fe(O)OH (13.44% w/v ofFe(O)OH particles, Noah Technologies Corporation, San Antonio, Tex.),372.9 g is suspended in 1899 mL of water and added to the deferoxaminewith constant stirring. The resulting suspension is heated to 60° C. forbetween 1 and 24 hours and the pH adjusted periodically to between 6.5and 7.9 by addition of 0.10N NaOH. Formation the ferrioxamine complex isevidenced by development of an intense deep reddish-brown color to thesolution. Stoichiometric chelation of Fe(III) with deferoxamine isconfirmed by in-process UV-Visible absorbance spectroscopy at 430 nm,against stoichiometrically chelated ferrioxamine standards. The batchsolution is cooled to room temperature and centrifuged at 4500 rpm(≈2500 g) for 15 minutes to remove any unreacted or aggregated Fe(O)OH.This final batch volume is adjusted as desired, typically to a finalvolume of 2600 mL. Any remaining trace amounts of unreacted Fe(O)OH areremoved and the solution also made aseptic, by passing the supernatantthrough a 0.22 μm Millipore GV-type filter in a Class 100 laminar flowhood. The resulting batch is stored at 4° C. in an autoclaved, sealedglass container until further use (see Examples below). The finalconcentration of ferrioxamine (DFe) is determined once again byUV-Visible absorbance spectrophotometry at 430 nm.

EXAMPLE 3 Preparation of the Basic Amine Chelator:Diethylenetriaminepentaacetate-Lysine (DTPA-Lys)

DTPA, 500 mg, is dissolved in 20 mL of pharmaceutical-grade water andheated to 60° C. L-Lysine hydrochloride powder, 931 mg, is added withconstant stirring until dissolved. Alternatively,N-epsilon-t-BOC-L-lysine can be used to prevent reaction of thecarbodiimide intermediate at the lysine epsilon amino group (see below),and when used, is dissolved in dimethylformamide:water (50:50, w/v). Thesolution pH is adjusted to 4.75 by addition of 0.1N HCl. Thewater-soluble carbodiimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC), 732.5 g, isdissolved in 2 mL water and its pH also adjusted as above. This EDCsolution is added dropwise to the DTPA+lysine solution mixture (above)over 1 hour at 22° C. with constant stirring and periodic adjustment ofpH to 4.75, and the reaction allowed to proceed to completion over 2more hours. When N-epsilon-t-BOC-L-lysine is used (see above), theN-epsilon-t-BOC group is hydrolyzed at this step, by acidification withhydrochloric acid to a pH of between 1.0 and 2.0, and stirring for 30-60min. The pH is readjusted to 4.75 as needed, and the reaction solutionis concentrated down to 5 mL by rotary evaporation at 60° C., and theDTPA-lysine (DTPA-Lys) derivative is precipitated by addition of 3volumes of ethanol. Note: under these conditions, the ethanol:waterratio used, maintains the solubility of all individual substrates(above). The resulting precipitate is harvested by centrifugation at2,500×g, washed with ethanol, re-centrifuged, and dried over a stream ofdry nitrogen. Covalent conjugation of lysine to DTPA is confirmed byinfrared (IR) spectroscopy. The resulting reaction product has a faintyellow color.

EXAMPLE 4 Preparation of the Gadolinium(III) Metal Chelate of DTPA-Lys:gadolinium:DTPA-Lys Gd(III):DTPA-Lys!

The gadolinium(III) chelate of DTPA-Lys, namely Gd(III):DTPA-Lys, isprepared by dissolving a known quantity of DTPA-Lys in water and addinga stock solution of gadolinium chloride, prepared at 0.1-1.0M, asneeded, until a stoichiometric quantity of Gd(III) has been added. ThepH is adjusted to 7.0 by addition of 1.0N NaOH. Alternatively,gadolinium oxide can be added and the reaction mixture stirred for 24hours. In the case of gadolinium oxide, neutralization with 1.0N NaOH isnot needed. Each batch of Lys-DTPA conjugate is pre-titrated and thefinal chelation product checked for stoichiometric addition of Gd(III),using a standard xylenol orange titration method Lyle et al. (1963)!,and further confirmed by quantitative ICP atomic absorption spectroscopyfor gadolinium. The resulting Gd(III):DTPA-Lys is precipitated byaddition of ethanol (3 volumes per volume of water), and the precipitatecollected by centrifugation. This precipitate is rewashed with ethanoland centrifuged (as above), washed with acetone plus centrifuged, andthe collected precipitate dried over a stream of dry nitrogen. Theresulting product continues to have the same faint yellow color as notedin Example 3.

EXAMPLE 5 Preparation of Paired-ion Agents of Ferrioxamine Bound toDermatan Sulfate Carriers; and Ferrioxamine to Depolymerized DermatanSulfate Carrier

Ferrioxamine:dermatan sulfate paired-ion agents are prepared by mixingappropriate ratios of the water solutions of ferrioxamine (see Example2, above) with either: (a) dermatan sulfate of modal MW betweenapproximately 5,000 daltons and 45,000 daltons (Opocrin, S.p.A., Modena,Italy; and Scientific Protein Laboratories, Waunake, Wis.); or (b)depolymerized dermatan sulfate of modal MW between approximately 2,000daltons and 5,000 daltons (Opocrin S.p.A., Modena, Italy). A range ofratios of ferrioxamine to dermatan sulfate are prepared between a low of1:99 (wt %) of ferrioxamine:dermatan sulfate or depolymerized dermatansulfate; and a high of 30:70 (wt %) of ferrioxamine: dermatan sulfate ordepolymerized dermatan sulfate). Using 0.1 to 1.0N NaOH, the pH of themixture is adjusted to between 5.5 and 8, the mixture is stirredcontinuously for 0.5 to 72 hours and the pH re-adjusted between 5.5 and8, and typically to 7.5. This ferrioxamine:dermatan mixture is passedthrough a 0.22 μm filter to remove any residual insoluble iron oxidesand hydroxides, and to render the liquid agent aseptic. The asepticagent is stored either as a liquid at 4° C., or as a lyophilized powder(see below). Further processing is carried out on the liquid, by fillinginto glass vials and autoclaving at 120° C. for 15 minutes.Alternatively, further processing is carried out on the liquid byfilling into glass vials, freezing at -50° C., and lyophilization togive an aseptic lyophilized powder. The lyophilized vials arereconstituted by adding sterile water and hand mixing for 1 to 5minutes, to give a reconstituted liquid of desired concentration whichis ready for injection. The resulting concentrations of ferrioxamine anddermatan sulfate are measured and vial quantities confirmed by standardreverse-phase HPLC and macromolecular size exclusion HPLC methods,respectively.

Multiple batches of Ferrioxamine:Dermatan Sulfate Agent have beenprepared. In vitro test results for a representative batch are asfollows: ferrioxamine:dermatan sulfate ratio: 77.5:22.5 (w/w);solubility of agent, 550 mg/mL; water:octanol partition, 17,600(±2,750):1; concentration of ferrioxamine, 0.166 mmol/mL; concentrationof dermatan sulfate, 32 mg/mL; molecular weight of dermatan sulfate,MN=18,000 daltons; sulfate/carboxylate ratio of dermatan sulfate,1.0±0.15; ferrioxamine and dermatan purities, nominal ±10%; pH, 6.5-7.9;viscosity, 3.8-4.2 centipoise; osmolality, 475-525 mOsm/Kg; R1, 1.5-1.8mmol.sec!-1; oversized particles, within USP guidelines for small-volumeparenterals; Anticoagulant activity, less than 4.5 U.S.P. Units/mg(modified USP XXII assay); binding of ferrioxamine active to dermatancarrier, at least 92% retained (dialysis for 3 hours against 200volumes, 500 daltons molecular weight cutoff).

In vitro stability of Ferrioxamine:Dermatan Sulfate Agent underaccelerated conditions, indicate the following. (a) The liquid form isstable, by the preceding physicochemical and HPLC parameters for longerthan 6 months at 4° C., 22° C. and 40° C.; is slightly unstable at 2 to6 months at 60° C., and degrades significantly within 1 to 3 days at 80°C. (b) The liquid form can be autoclaved as above, with less than 3%degradation of ferrioxamine. (c) The lyophilized form is stable withrespect to all parameters (above), including oversized particles; and isprojected to be stable over storage periods of multiple years.

EXAMPLE 6 Preparation of Paired-ion Agents of Ferrioxamine Bound toHeparin

Ferrioxamine:dermatan sulfate paired-ion agents are prepared by mixingappropriate ratios of water solutions of ferrioxamine (as in Example 5,above) with (a) beef lung heparin of modal MW between approximately8,000 daltons; and (b) porcine heparin of modal MW between approximately10,000 daltons and 20,000 daltons. A range of ratios of ferrioxamine toheparin or heparin fragment are prepared between a low of 1:99 (wt/wt)of ferrioxamine:heparin or heparin fragment; and a high of 30:70 (wt offerrioxamine:fragment. Using 0.1 to 1.0N NaOH, the pH of the mixture isadjusted to between 5.5 and 8, the mixture is stirred continuously for0.5 to 72 hours and the pH re-adjusted between 5.5 and 8. Thisferrioxamine:heparin mixture is passed through a 0.22 μm filter toremove any residual insoluble iron oxides-hydroxides and render theliquid agent aseptic. The aseptic agent is stored at 4° C. As indicated,further processing is carried out by filling the aseptic liquid in glassvials, followed by freezing and lyophilizing, to render the agent as anaseptic lyophilized powder. The lyophilized vials are reconstituted byadding sterile water and hand mixing for 1 to 5 minutes, to give areconstituted liquid of desired concentration which is ready forinjection. The resulting concentrations of ferrioxamine and heparin aremeasured and vial quantities confirmed by standard reverse-phase HPLCand macromolecular size exclusion HPLC methods, respectively.

EXAMPLE 7 Preparation of Non-anticoagulant Heparin Carrier By GlycineDerivatization

The anticoagulant activity of heparin can be reduced to almostnegligible activity by derivatizing its carboxylate groups with glycineresidues as reported Danishefsky et al. (1971); Danishefsky et al.(1972)!. This non-anticoagulant heparin (Nac-heparin) can then beutilized as a modified glycosaminoglycan carrier. According to onepresent method of glycine conjugation, 0.75 g of heparin is weighed intoa 100 mL beaker and dissolved in 25 mL of pharmaceutical-grade water.Glycine, 0.75 g, is added and the pH of the resulting solution adjustedto 4.75 with 0.10N HCl. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimideHCl (EDC), 0.75 g, is weighed into a separate vial, solubilized byadding a minimum amount of water, and the pH adjusted to 4.75 with 0.10MHCl. Aliquots of the EDC solution are added to the mixture ofglycine-glycosaminoglycan over a one hour period. After each addition ofEDC, the pH is adjusted to maintained it at 4.75. After addition of allEDC, the reaction is allowed to proceed for an additional two hours withconstant stirring and periodic pH adjustment. The glycine-heparinconjugate (Gly-HEP) is then precipitated by addition of 3 volumes ofabsolute ethanol. The precipitate is collected by centrifugation at 4500rpm (≈2500×g) for 15 minutes; and washed three times with 20-mL aliquotsof ethanol with re-centrifugation.

EXAMPLE 8 Preparation of Paired-ion Agents of Ferrioxamine Bound ToGlycosaminoglycans, Modified and Derivatized Glycosaminoglycans of:heparan sulfate, non-anticoagulant heparin oversulfated dermatan sulfatechondroitin sulfate, oversulfated chondroitin sulfate and the bacterialSulfatoid, pentosan polysulfate

Ferrioxamine paired-ion agents are prepared with variousglycosaminoglycan carriers by mixing appropriate ratios of watersolutions of ferrioxamine (as in Example 5, above) with the followingglycosaminoglycans: (a) heparan sulfate of MN=8,500 daltons; (b)non-anticoagulant heparin SPL, ++ of MN=10,500 daltons; (c) oversulfateddermatan sulfate of MN=19,000 daltons; (d) chondroitin sulfate ofMN=23,400 daltons; (e) oversulfated chondroitin sulfate of MN=14,000daltons; and (f) pentosan polysulfate of MN=2,000 daltons. The ratios offerrioxamine to glycosaminoglycan and sulfatoid carriers are prepared togive a payload of 77.5:22.5% (w/w) of ferrioxamine to carrier!(adjusted) by a scaling factor of (mEq sulfates/mg of carrier asabove)/(mEq sulfates/mg of beef lung heparin*)!. Using 0.1 to 1.0N NaOH,the pH of the mixture is adjusted to between 5.5 and 8, the mixture isstirred continuously for 0.5 to 72 hours and the pH re-adjusted between5.5 and 8. This ferrioxamine:heparin mixture is passed through a 0.22 μmfilter to remove any residual insoluble iron oxides-hydroxides andrender the liquid agent aseptic. The aseptic agent is stored at 4° C. Asindicated, further processing is carried out by filling the asepticliquid in glass vials, followed by freezing and lyophilizing, to renderthe agent as an aseptic lyophilized powder. The lyophilized vials arereconstituted by adding sterile water and hand mixing for 1 to 5minutes, to give a reconstituted liquid of desired concentration whichis ready for injection. The resulting concentrations of ferrioxamine andheparin are measured and vial quantities confirmed by standardreverse-phase HPLC and macromolecular size exclusion HPLC methods,respectively.

Although not prepared in the present application, it is apparent that bycombining the teaching of the present Example with those of previousdisclosures 07/880,660, 07/803,595, and 07/642,033, ferrioxaminecomplexes can be similarly prepared with additional acidic saccharides,including sucrose octasulfate and sulfated cyclodextrins; withadditional glycosaminoglycans, including keratan sulfate andhyaluronate; and with additional sulfatoids, including the bacterialsulfatoid, dextran sulfate.

EXAMPLE 9 Preparation of Paired-ion Agents of Gd(III):DTPA-Lys Bound toDermatan Sulfate Carrier

Gd(III):DTPA-Lys:Dermatan Sulfate paired-ion agents are prepared bymixing the water solutions of Gd(III):DTPA-Lys with dermatan sulfate ofmodal MW between approximately 5,000 daltons and 45,000 daltons (as inExample 5, above), and in particular, dermatan sulfate of MN=18,000(Opocrin, S.p.A., Modena, Italy), to form a final solution ratio of77:30% (w/w) of the Gd(III):DTPA-Lys active to the Dermatan Sulfatecarrier. Several stable Agent variations of the resulting liquid havebeen prepared, wherein the concentration of Gd(III):DTPA-Lys ranges from0.166 to 0.415 mmol/mL, and the respective concentration of dermatansulfate ranges from 35 to 87.5 mg/ml.

EXAMPLE 10 Preparation of a Basic Iron-porphine Chelate; and Paired-ionBinding to Heparin

The soluble, tetra-basic porphine,5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H-23-Hporphine, 40 mg as thetetra-p-tosylate salt, is refluxed with Fe(II) chloride, 30 mg, for 2hours in 20 mL of dimethylformamide. Evidence of iron complexation isobserved in the form of a red to dark green color. Solvent was removedby evaporation, the solid product dissolved in water. The pH is adjustedto 7.5 to insolubilize excess ferric iron, followed by filtration of theiron-porphine product. A 2 mg/mL solution of iron-porphine complex andca. 100% product yield is confirmed by inductively coupled plasma atomicabsorption. A comparable reaction in water gives ca. 70% yield.

This iron-porphine complex is added to beef lung heparin dissolved inwater, ca. 8 Kd, at ratios ranging from 1:20 to 20:1(iron-porphine:heparin). This resulted in clear solutions withoutprecipitates. Binding of iron-porphine to heparin is nearly 100% asevaluated by dialysis against water for 16 hours, using bags withmolecular weight cutoffs of 3.5 Kd and 12 Kd. Iron-porphine alone isnearly completely dialyzed. UV-Visible spectrophotometric titrationindicates maximum binding occurs at a molar ratio of 18:1(iron-porphine:heparin). Since the beef lung heparin used is known tohave approximately 18 available strongly acidic (sulfate) groups permole (and per heparin chain), these results indicate strong ionicinteraction and stable (to dialysis) binding of the basic tetraamineporphine complex to the sulfate groups of heparin.

EXAMPLE 11 Preparation of a Basic Triethylenetetraamine-iron Chelate;and Paired-ion Binding to Heparin and Sucrose Octasulfate

Soluble complexes of triethylenetetraamine and iron(III) are formed bydissolving 1.0 g of triethylenetetraamine.2HCl (Syprine™) (Merck, WestPoint, Pa.) in water and adding a 1:1 mole ratio of iron chloride underacidic conditions (pH=2) to give a clear yellow solution. Using 0.1NNaOH, the pH is adjusted to 6.8, giving a red solution indicative ofiron complexation. This solution develops a feathery red precipitate,indicative of intermolecular aggregation of theiron-triethylenetetraamine complex.

(a) To this resulting aqueous dispersion of complex is added beef lungheparin, to give final complex-to-heparin ratios of between 95:5 and5:95 (by weight). At a ratio of 65:35 (complex:heparin) and higherratios of heparin, heparin completely solubilizes the complex. Thisapparent solubilization is indicative of paired-ion binding betweentriethylenetetraamine-iron and heparin.

(b) To the aqueous dispersion of triethylenetetraamine-iron complex isadded sucrose octasulfate (SOS), to give final complex-to-SOS ratios ofbetween 95:5 and 5:95 (by weight). At a ratio of 65:35 (complex:SOS) andhigher ratios of SOS, SOS causes the dispersion to become very muchfiner, indicative of paired-ion binding betweentriethylenetetraamine-iron complex and SOS. The absence of completeclarification of this SOS paired-ion system relative to that withheparin (above), is due to the much higher density of sulfates on SOSrelative to heparin, which confers substantially increasedintermolecular hydrogen bonding on the SOS system.

Although not directly exemplified, it will be apparent that polyamineswith the homologous series C_(x) H_(x+y) N_(x-z), which also form stablecomplexes with Iron(III), can also be used in place oftriethylenetetraamine-iron complex and SOS in the present invention.

Preparation of Covalent Conjugates of Deferoxamine GlycosaminoglycanCarriers

Substrates with electrophilic amine groups may be covalently conjugatedreagents to nucleophilic carboxylate groups of acidic carriers, acidicsaccharides and acidic glycosaminoglycans as reported Danishefsky et al.(1971); Danishefsky et al. (1972); Janoki et al. 1983); Axen (1974);Bartling et al. (1974); Lin et al. (1975)!. The coupling reagentsdescribed in these references activate carboxylate groups towardnucleophilic attack. The mechanism involves formation of an activatedintermediate resulting from reaction of the coupling reagent with thecarboxylate residues on the carrier. The intermediate undergoesnucleophilic attack, typically by an amine functional group. Thisresults in formation of a stable covalent conjugate, typically via anamide bond between the active and the carrier. Examples 12, 13, and 14(below) describe the synthesis of ferrioxamine-heparin covalentconjugates, wherein the ferrioxamine is covalently bound to heparin viathree different coupling reagents.

EXAMPLE 12 Preparation of a Covalent Ferrioxamine-Heparin Conjugate by1-ethyl-3-(3-dimethylaminopropyl) Carbodiimide (EDC) Linkage

Aqueous ferrioxamine, 2.0 g, as prepared in Example 1, is adjusted to pH4.75 by addition of 0.10M HCl. Beef-lung heparin (Hepar-Kabi-Pharmacia,Franklin, Ohio), 0.75 g, is dissolved 5.0 mL of pharmaceutical-gradewater and added to the ferrioxamine with constant stirring. The pH ofthe resulting solution is readjusted to 4.75 with 0.10M HCl. Thewater-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC), 2 g, is weighed into a scintillation vial,solubilized in a minimum amount of water, and the pH adjusted to 4.75with 0.10M HCl. Aliquots of EDC solution are pipetted into the mixtureof ferrioxamine-heparin over a one hour period. After each addition ofEDC the 0.10M HCl is added to maintain the pH at 4.75. After addition ofall EDC, the reaction is allowed to proceed for an additional two hourswith constant stirring. The ferrioxamine-heparin conjugate isprecipitated by addition of 3 volumes of absolute ethanol. Thisprecipitate is collected by centrifugation at 4500 rpm (≈2500×g) for 15minutes and washed three times with 20 mL aliquots of ethanol pluscentrifugation. The complex is further purified by redissolving in waterand reprecipitating with 3 volumes of ethanol plus centrifugation. Thefinal product is collected and dried over nitrogen. Ferrioxaminederivatization of heparin is confirmed by UV-visible absorbancespectroscopy of the ferrioxamine chelate at 430 nm and heparin analysisby size-exclusion HPLC chromatography.

EXAMPLE 13 Preparation of a Covalent Ferrioxamine-Heparin Conjugate byN-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) Linkage

Beef-lung heparin (Hepar-Kabi-Pharmacia, Franklin, Ohio), 0.50 g, isweighed into a 3-necked 100 mL round bottom flask fitted with an inletand outlet for N₂ purge. Anhydrous dimethylformamide (DMF), 20 mL, isadded with constant stirring and the resulting suspension warmed to 50°C. under a constant flow of nitrogen. A 30 mole excess (≈463.7 mg) ofN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is added and theresulting suspension stirred at 50° C. for 3 hours. The activatedEEDQ-activated heparin is collected by centrifugation at 4500 rpm(≈2500×g) for 10 minutes. The pellet is washed repeatedly with anhydrousDMF and then 3 times with acetone. The activated intermediate is driedunder a stream of nitrogen.

An aliquot of ferrioxamine solution containing 766.3 mg of the ironcomplex, as prepared in Example 1, is pipetted into a 50 mL beaker anddiluted to 25 mL with anhydrous DMF. In a separate 50 mL beaker, a knownamount of EEDQ-activated heparin is suspended in 50 mL of anhydrous DMFwith constant stirring. The DMF solution of ferrioxamine is pipettedslowly into the EEDQ-heparin suspension over a 5 minute period. Theresulting suspension is stirred continuously for 3 hours at 40° C. Aftercooling to room temperature, the final product is collected bycentrifugation, washed three times with anhydrous DMF, washed threetimes with acetone, and dried under nitrogen. Confirmation of conjugateformation is performed as in Example 12.

EXAMPLE 14 Preparation of a Covalent Ferrioxamine-Heparin Conjugate byCarbonyldiimidazole (CDI) Linkage

An activated intermediate of beef-lung heparin (Hepar-Kabi-Pharmacia,Franklin, Ohio) is prepared by weighing 3.0 g of heparin into a 50 mLround bottom flask and adding 25 mL of anhydrous dimethylformamide (DMF)with constant stirring. Carbonyldiimidazole (CDI), 608.1 mg, (10 moleexcess relative to heparin) is weighed into a separate vial anddissolved in 20 mL of anhydrous DMF. The DMF solution of CDI is added tothe DMF-heparin suspension and stirred at 30° C. for one hour. TheCDI-activated heparin is collected by centrifugation, washed repeatedlywith acetone to remove unreacted CDI and residual DMF, and dried undernitrogen.

The deferoxamine-heparin conjugate is prepared by weighing 1.0 g of theCDI-activated heparin into a 50 mL round bottom flask and suspendingthis in 25 mL of anhydrous DMF. Deferoxamine, 250 mg, prepared as inExample 1, is weighed into a separate round bottom flask and dissolvedin 20 mL of anhydrous DMF. The deferoxamine free base solution is addedslowly to the CDI-heparin suspension and stirred continuously for 16hours at 75° C. The deferoxamine-heparin conjugate is collected bycentrifugation at 4500 rpm (≈2500×g) for 15 minutes, washed repeatedlywith anhydrous DMF, washed repeatedly with acetone, and dried undernitrogen. The resulting product is dissolved in water, and itsconcentration determined by UV-Visible spectroscopy. A stoichiometricquantity of aqueous FeCl₃ is added and the resulting solution adjustedgradually to pH 6.5 and stirred for 2 hours. This results in a deepbrown-red product. This ferrioxamine-heparin conjugate is separated fromany residual substrates and intermediates by dialysis through a 2,000 MWcutoff bag against 150 volumes of water. The retentate is collected andconcentrated by rotary evaporation. Confirmation of derivatization isperformed as in Examples 12 and 13.

EXAMPLE 15 Preparation of a CovalentHeparin-Diethylenetriaminepentaacetate Conjugate (DTPA-heparin)

DTPA-functionalized carriers are prepared in aqueous media from thereaction of diethylenetriaminepentaacetic dianhydride (cDTPAA;Calbiochem-Bhering Corp.) and a molecule containing a nucleophilicfunctional group. Beef-lung heparin (Hepar-Kabi-Pharmacia, Franklin,Ohio), 1.5 g, is dissolved in 75.0 mL of 0.05M HEPES buffer and the pHadjusted to 7.0 with 0.10M NaOH. cDTPAA, 4.5 g (≈100 mole excessrelative to heparin), is weighed out and divided into 20 equal (225 mg)aliquots. An aliquot of cDTPAA is added to the heparin solution every3-5 minutes until all cDTPAA has been added. The pH of the solution ismonitored continuously throughout cDTPAA addition and maintained at pH7.0 with 0.10M NaOH. After addition of the last aliquot of cDTPAA, thesolution is stirred for an additional 30 minutes. The DTPA-heparinsolution is dialyzed through 1000 MW bags against 150 volumes to removenon-conjugated DTPA. The resulting conjugate is concentrated bynitrogen-evaporation at 37° C. and stored at 4° C.

EXAMPLE 16 Preparation of Gadolinium(III) and Iron(III) Chelates ofDTPA-heparin Covalent Conjugate

The DTPA-heparin conjugate of Example 15 is further prepared in the formof paramagnetic metal chelates of the DTPA group with gadolinium(III) orFe(III), by pipetting the required volume of DTPA-heparin into a 125 mLErlenmeyer flask, adding a 1.5-to-10 mole excess of the paramagneticmetal ion oxide, as Gd₂ O₃ or Fe(O)OH, and stirring for 24 to 36 hoursat 37° C. to obtain solubilization of the metal oxides sufficient forcomplete occupancy of the DTPA groups. The residual metal oxides areprecipitated by centrifugation at 4500 rpm (≈2500 g), and the productseparated from unreacted metal oxides by filtration through a Millipore0.22 μm GV-type filter, followed by dialysis against 150 volumes. Theconcentrations of chelated metal ion and heparin are determined byinductively coupled plasma (ICP) and size-exclusion HPLC, respectively.In the case of Gd(III), stoichiometric chelation is also confirmed bystandard xylenol orange titration Lyle et al. (1963)!.

EXAMPLE 17 Toxicity Studies of Ferrioxamine:Dermatan Sulfate

Acute intravenous Toxicity Studies with 14-day recovery and necropsy areperformed in male and female rats and male and female dogs. At standardi.v. injection rates of 0.075 mmol/Kg/min., significant signs generallyoccur only after 5-12.5 1 times the effective imaging dose of 0.155mmol/Kg. The LD50 is much greater than 4.5 mmol/Kg and is limited bytechnical aspects of tail-vein infusion. At this rate, some rats can beinfused with 10 mmol/Kg without untoward effects. At an artificiallyaccelerated i.v. injection rate of 0.080 mmol/Kg, deaths in rats can beobtained, and the LD50 is between 2.5 and 3.0 mmol/Kg. Terminal necropsyreveals no abnormalities in any rats after i.v. injection of 2.2, 3.0and 4.5 mmol/Kg (n=5 males and 6 females per dose level).

A pyramid acute i.v. toxicity study is performed in dogs at escalatingdoses of 0.5, 1.2 and 2.25 mmol/Kg and an infusion rate of 0.012mmol/Kg/min in protocol studies. An acute symptom complex of hypotensioncan be obtained, which is minimal and reversible. No deaths occurred andterminal necropsy at 14 days revealed no abnormalities (n=2 males and 2females, all administered each of the three dose levels, with a 72-hourrest interval).

EXAMPLE 18 Ferrioxamine:Dermatan Sulfate Selective Contrast Agent: MRIImaging of Lactating Breast Adenocarcinomas in Syngeneic Fisher 344Female Rats

As shown in FIGS. 2A-4d, T1-weighted MRI images (TR/TE-800/45 and550/23) are performed at 1.0 and 1.5 Tesla, before (Pre) and after(Post) intravenous (i.v.) injection of Ferrioxamine:Dermatan SulfateSelective Paramagnetic Contrast Agent (Example 5), at a Ferrioxaminedose of 0.155 mmol/Kg into Fisher 344 female rats, with syngeneic breastadenocarcinomas inoculated by trocar into the livers, such that tumordiameters at the time of imaging are between 1.0 cm and 2.5 cm. Tumorsare not conspicuous on standard T1-weighted Precontrast images.Following injection of Ferrioxamine:Dermatan Sulfate Agent, the tumors(a) become rapidly and markedly enhanced at an early postinjection time(7 mins) (FIGS. 2A-B); (b) display very sharp tumor boundaries againstsurrounding liver (FIGS. 2A-B and 4A-D), and discretely demarcated,darker central region of tumor necrosis (FIGS. 2A-B) (allowing tumorperfusion and function to be spatially resolved and assessed withindifferent, very small anatomical subregions); (c) exhibit sustainedcontrast for longer than 64 minutes postinjection (MPI) (FIGS. 4A-D, MRIimages; FIG. 5, quantitative region-of-interest, ROI, analysis) withcontinued very well defined tumor borders at prolonged imagingintervals. MRI images and microwave augmented iron stains of the freshlyexcised, 7 MPI tumors, indicate that tumor-site localization of theFerrioxamine active occurs only when it is bound (non-covalently) tocarrier (FIGS. 2A-B and 4A-D) and not when administered in free form(Active alone) (FIGS. 3A-B). As shown in FIGS. 8A-C, lung metastases ofthe liver tumor are rapidly and sensitively enhanced in very small 2-mmto 3-mm nodules at an early post-contrast interval; and this enhancementof the tumor at lung sites is also sustained for a prolonged period withhigh sensitivity plus retention of very sharp tumor boundaries againstnormal lung. The sustained intervals shown in FIGS. 8A-C are much longerthan those typically reported for Gd:DTPA dimeglumine contrastenhancement at body organ sites.

EXAMPLE 19 Ferrioxamine:Dermatan Sulfate Selective Contrast Agent: MRIImaging of Prostate AT-1 Carcinomas in Syngeneic Copenhagen Rats andComparison with Gd(III)DTPA

As shown in FIGS. 9A-E and 10A-E, T1-weighted MRI images (TR/TE-250/80)performed at 4.7 Tesla, before (Pre) and after (Post) intravenous (i.v.)injection of Ferrioxamine:Dermatan Sulfate Selective ParamagneticContrast Agent prepared as in Examples 2 and 5, and injected i.v. at anIron(III) dose of 0.155 mmol/Kg (FIGS. 9A-E); compared to GadoliniumDTPA dimeglumine, injected i.v. at a Gd(III) dose of 0.100 mmol/Kg(FIGS. 10A-E); each of these agents being administered to Copenhagenrats with syngeneic AT-1 prostate adenocarcinomas inoculated intopreviously prepared skin pouches Hahn, et al. !, such that tumordiameters at the time of imaging are between 1.0 cm and 2.5 cm.Ferrioxamine:Dermatan Sulfate produces a rapid large enhancement of theOuter Rim of tumor and also of the Vascular Array which fans out fromthe tumor pedicle which carries a high majority of the tumorvasculature. Sustained contrast and delineation of these elementsremains present through kinetic time points of 40 minutes. Bycomparison, following Gd:DTPA dimeglumine, the outer rim is not welldelineated, even at the earliest post-contrast interval (7 MPI). Markedearly contrast fading occurs overall in the tumor at 20 MPI, and someagent sequesters in the central, poorly perfused (cystic) regions oftumor (as is typically reported for Gd:DTPA when used for imaging atbody sites). At 40 MPI, enhancement reverts to essentially backgroundlevels, and at 60 MPI, there is no residual contrast, except for centralcystic regions.

EXAMPLE 20 MRI Contrast Enhancement of Acute Dog Myocardial Infarcts byFerrioxamine:Dermatan Sulfate

As shown in FIGS. 11A-D, T1-weighted MRI ECG-gated cardiovascular imagesare performed at 0.5 Tesla, before (Pre) and after (Post) rapidintravenous (i.v.) infusion of Ferrioxamine:Dermatan Sulfate SelectiveParamagnetic Contrast Agent injected i.v. at an Iron(III) dose of 0.155mmol/Kg into German Shepherd dogs with acute, 90-min myocardial infarcts(ligature of proximal left anterior descending coronary artery) followedby reperfusion for ca. 90 minutes prior to contrast agent infusion. At 7MPI, Ferrioxamine:Dermatan gives strong enhancement of the infarct zone,and in particular distinguishes the outer boundary of the infarct, whichrepresents the putative marginal zone of the infarct amenable topotential recovery, from the central darker region, which represents theputative irreversible central infarct. Sustained strong enhancement andzonal demarcation is present through 40 MPI. Ferrioxamine injectedwithout carrier at 0.155 mmol/Kg, gives no detectible enhancement. Inthese studies, infarct sizes and positions are documented by double dyeinfusion performed immediately after MRI imaging.

EXAMPLE 21 Comparison of MRI Tumor-imaging Potency In Vivo withFerrioxamine Active Bound to Various Sulfated Glycosaminoglycans

Based on low anticoagulant activity, safety and projectedsite-localization potential, certain alternative glycosaminoglycancarriers and certain alternative physical forms of the resultingSelective MRI Contrast Agents are compared for their relative in vivopotencies of carrier-mediated tumor localization of bound Ferrioxamine.Because of its high spatial resolution and capacity to detect subtlequantitative differences in agent localization, the AT-1 prostate tumormodel of Example 19 is used.

                  TABLE 2                                                         ______________________________________                                                                     Concentra-                                                                            Relative                                                    Form      tion    Potency                                  FIG.               (Liquid/  (metal, (scale of                                NO.    Agent       Lyo)      mmol/mL)                                                                              1-6)                                     ______________________________________                                        12A-D  Ferrioxamine                                                                              Lyo       0.415   3.5                                             Dermatan-SO.sub.3.sup.-                                                13A-D  Gd:DTPA-Lys Liquid    0.415   6                                               Dermatan-SO.sub.3.sup.-                                                14A-D  Ferrioxamine                                                                              Lyo       0.332   4.5                                             Oversulfated                                                                  Dermatan-SO.sub.3.sup.-                                                15A-D  Ferrioxamine                                                                              Lyo       0.332   5                                               Oversulfated                                                                  Chondroitin-SO.sub.3.sup.-                                             16A-D  Ferrioxamine                                                                              Lyo       0.332   3.5                                             Heparan Sulfate                                                        ______________________________________                                    

Carriers of shorter chain length than the glycosaminoglycans, namelypentosan polysulfate, are found to be less potent (typically only 2/6 onthe scale above) and remain at the tumor site for intervals of less thanabout 20 minutes, whereas the GAGs shown in the table above, are muchmore potent and have considerably longer tumor site localizationintervals. In comparing these carriers, there is a slight-to-moderatetrend towards increased carrier potency based on carrier sulfate chargedensity.

Lyo=Lyophilized powder form

SO₃ ⁻ =Sulfate (e.g. SO₃ ⁻ =dermatan sulfate)

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it will be apparent to those of skillin the art that variations may be applied to the composition, methodsand in the steps or in the sequence of steps of the method describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

The following references are incorporated in pertinent part by referenceherein for the reasons cited above.

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What is claimed is:
 1. An agent comprising at least about 15 weight percent chelator for paramagnetic metal ions, and a sulfated oligosaccharide, sulfated polysaccharide or glycosaminoglycan carrier, said chelator having a cationic group and being bound to said carrier by non-covalent binding.
 2. The agent of claim 1 wherein said chelator has a net positive ionic charge at physiological pH.
 3. The agent of claim 1 further comprising a chelated paramagnetic metal ion.
 4. The agent of claim 2 further comprising a chelated paramagnetic metal ion.
 5. The agent of claim 1 further comprising a chelated metal ion selected from the group consisting of iron, manganese, chromium, copper, nickel, gadolinium, erbium, europium, dysprosium and holmium.
 6. The agent of claim 1 wherein said carrier is a sulfated glycosaminoglycan or sulfatoid.
 7. The agent of claim 1, wherein the carrier is heparin, desulfated heparin, glycine-conjugated heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate, pentosan polysulflate or dextran sulfate.
 8. The agent of claim 1, wherein said chelator has a formation constant for paramagnetic metal ions of at least about 10¹⁴.
 9. The agent of claim 1, wherein said chelator is a chelator of iron ions.
 10. The agent of claim 1, wherein said chelator is a hydroxamate.
 11. The agent of claim 3, wherein said chelator together with said metal ion is ferrichrome, ferrioxamine, enterobactin, ferrimycobactin or ferrichrysin.
 12. The agent of claim 1, wherein said chelator is deferoxamine.
 13. The agent of claim 1, wherein said chelator is deferoxamine, said carrier is heparin, or a heparin fragment and the agent further comprises iron(III).
 14. The agent of claim 1, wherein said chelator is deferoxamine and said carrier is dermatan sulfate or a dermatan sulfate fragment.
 15. The agent of claim 14, wherein said agent further comprises chelated iron(III).
 16. An agent for magnetic resonance imaging comprising deferoxamine non-covalently bound to a carrier selected from the group consisting of heparin, heparan sulfate, dermatan sulfate and chondroitin sulfate.
 17. The agent of claim 16, further comprising a paramagnetic metal ion.
 18. The agent of claim 1, wherein said chelator is a porphine, porphyrin, sapphyrin or texaphyrin.
 19. The agent of claim 18 further defined as comprising a paramagnetic metal ion.
 20. The agent of claim 18, further defined as comprising an iron ion or a gadolinium ion.
 21. The agent of claim 1, wherein said chelator is 5,10,15,20-Tetrakis(1-methyl-4-pyridyl)-21H,23-porphine, said carrier is heparin and the agent further comprises a chelated iron ion.
 22. The agent of claim 1, wherein said chelator is a polyaminocarboxylate or macrocyclic.
 23. The agent of claim 22, further defined as comprising a paramagnetic metal ion.
 24. The agent of claim 22, wherein said chelator is an amine derivative of diethylenetriaminetetraacetate or is a derivative of diethylenetriaminetetraacetate having a net positive charge at physiological pH.
 25. The agent of claim 22, wherein said chelator is an amine derivative of 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetate (DOTA) or is a derivative of 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetate having a net positive charge at physiological pH.
 26. The agent of claim 1, wherein said carrier is defined further as being complementary to endothelial determinants selectively induced at disease sites.
 27. An agent for in vivo magnetic resonance imaging, the agent comprising a chelator having a net positive charge at physiological pH, a chelated metal ion and a sulfated hydrophilic carrier, said chelator/metal ion chelate being bound by non-covalent binding at a weight percentage of at least about 15% to said carrier, and said carrier being selected from the group consisting of heparin, desulfated heparin, glycine-conjugated heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate, pentosan polysulfate and dextran sulfate.
 28. An agent for enhancing body imaging, the agent comprising diethylenetriaminepentaacetate-lysine, chelated Gd(III) and a glycosaminoglycan carrier non-covalently bound to said diethylenetriaminepentaacetate-lysine.
 29. An agent for enhancing body imaging, the agent comprising DOTA-lysine, chelated Gd(III) and a glycosaminoglycan carrier non-covalently bound to said 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetate-lysine (DOTA-lysine).
 30. An agent for magnetic resonance imaging comprising ferrioxamine bound by non-covalent binding to dermatan sulfate.
 31. The agent of claims 1, 16, 27, 28 or 29 defined further as being in a combination with at least one of a buffer, saccharide, sulfated saccharide, or salt, to produce an osmotic strength suitable for parenteral administration, and as being an aqueous solution or a lyophilized or dry preparation suitable for aqueous reconstitution having the desired osmotic strength, and wherein said agent is aseptic or sterile.
 32. A method of enhancing magnetic resonance images or spectra in vertebrate animals comprising administering to said animal an effective amount of the agent of claim
 4. 33. A method of enhancing in vivo images arising from induced magnetic resonance signals, comprising the steps of:(a) administering to a subject an effective amount of the agent of claim 1; (b) exposing the subject to a magnetic field and radiofrequency pulse; and (c) acquiring an induced magnetic resonance signal to obtain a contrast effect. 